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Analysis of <t>TcAUK1</t> overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
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Images

1) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

2) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

3) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

4) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

5) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

6) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

7) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

8) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

9) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

10) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

11) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

12) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

13) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

14) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

15) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

16) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

17) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

18) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

19) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

20) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

21) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

22) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

23) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

24) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

25) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

26) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

27) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

28) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

29) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

30) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

31) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

32) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

33) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

34) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

35) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

36) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

37) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

38) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

39) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

40) Product Images from "Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication"

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0007256

Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
Figure Legend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

Techniques Used: Isolation, Southern Blot, Transgenic Assay

TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.
Figure Legend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

Techniques Used: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).
Figure Legend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.
Figure Legend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

Techniques Used: Isolation, Infection, Staining

Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.
Figure Legend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

Techniques Used: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).
Figure Legend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

Techniques Used: Staining

Related Articles

Staining:

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication
Article Snippet: .. Furthermore, TcAUK1 adopted the same configuration as the mitotic spindle, as shown by microtubule staining, revealing a close association of TcAUK1 with this structure. .. To study TcAUK1 localization in the other forms of T . cruzi , trypomastigote and amastigote forms were collected from culture supernatant of infected Vero cells and subjected to immunostaining with TcAUK1 antiserum ( ).

Southern Blot:

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication
Article Snippet: .. Probes for TcAUK1 and TcAUK2 were included in this experiment to confirm the Southern blot results. .. While only one band was observed for TcAUK1 and TcAUK2, supporting results that indicated these are single copy genes, two bands were detected for TcAUK3, confirming the presence of more than one CDS located in different chromosomes.

Over Expression:

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication
Article Snippet: .. Considering that TcAUK1 seems to be involved in mitosis, we hypothesized its overexpression could alter cell cycle progression, modifying the culture growth rate. ..

other:

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication
Article Snippet: When TcAUK1 or TcAUK2 probes were used, the results confirm that both are single copy genes.

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication
Article Snippet: During mitosis (2F2K2N), when both kinetoplasts are moving in opposite directions, TcAUK1 was detected inside the nucleus.

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication
Article Snippet: In amastigotes, TcAUK1 appeared as a single focus close to the kinetoplast, whereas in trypomastigotes no TcAUK1 signal was detected.

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication
Article Snippet: Surprisingly, here TcAUK1 was detected not only in amastigotes but also in trypomastigotes and in both cases located inside the parasite nucleus ( , 2 days).

Expressing:

Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication
Article Snippet: .. After confirming the expression of TcAUK1 in the amastigote, trypomastigote and epimastigote stages , we next aimed to study its localization during epimastigotes cell cycle. .. By immunofluorescence, we detected TcAUK1 at two discrete cell cycle stage-dependent subcellular localizations: during interphase, it is localized at the extremes of the kinetoplast while in mitosis it resides inside the nucleus, associated with the mitotic spindle ( ).

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    GenScript tcauk1
    Analysis of <t>TcAUK1</t> overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p
    Tcauk1, supplied by GenScript, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

    doi: 10.1371/journal.pntd.0007256

    Figure Lengend Snippet: Analysis of TcAUK1 overexpressing epimastigotes. (A) Isolated DNA from pTREX and pTREX-TcAUK1 epimastigotes was treated with different restriction endonucleases and analyzed by Southern blot to confirm the extra copy of TcAUK1 in the genome of transgenic cells. (B) Immunoblot against TcAUK1 protein in pTREX and pTREX-TcAUK1 cells extracts. The β-Tubulin protein was used as loading control. (C) pTREX and clonal pTREX-TcAUK1 epimastigotes were cultivated and monitored for cell growth every day. Cell number was plotted in a logarithmic scale and the presented data is a mean ± s.d. of three independent cell cultures. Cells duplication time in each independent culture was calculated and a Paired T test (p

    Article Snippet: When TcAUK1 or TcAUK2 probes were used, the results confirm that both are single copy genes.

    Techniques: Isolation, Southern Blot, Transgenic Assay

    TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

    doi: 10.1371/journal.pntd.0007256

    Figure Lengend Snippet: TcAUK1 localization in epimastigote forms. (A) Epimastigote forms were cell cycle synchronized with HU and the indicated cell cycle phases confirmed by flow cytometry. TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI). (B) TcAUK1 coding sequence was cloned into pTEXeGFP expression vector and epimastigotes were transfected. Shortly after transfection (24–48 hs), the localization of the fusion protein was evaluated by fluorescence microscopy.

    Article Snippet: When TcAUK1 or TcAUK2 probes were used, the results confirm that both are single copy genes.

    Techniques: Flow Cytometry, Cytometry, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transfection, Fluorescence, Microscopy

    Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

    doi: 10.1371/journal.pntd.0007256

    Figure Lengend Snippet: Expression of TcAUKs genes. (A) RT-PCR of the three TcAUKs and Actin (housekeeping control) in epimastigotes (E), trypomastigotes (T) and amastigotes (A) of T . cruzi . (-): PCR negative control. I and II indicate the specific amplification products of similar length obtained for TcAUK3 (B) Nucleotide sequence of the 5´ Untranslated Region of each TcAUK mRNA. The Spliced Leader sequence and the starting ATG codon followed by the initial sequence of the genes (CDS) are indicated. I and II reference to the bands indicated in (A). Underlined in TcAUK3 is the region of the 5´ UTR absent in one of the two amplification products. (C) Immunoblot of TcAUK1 in total cell extracts from Epimastigotes (E), Trypomastigotes (T) and Amastigotes (A).

    Article Snippet: When TcAUK1 or TcAUK2 probes were used, the results confirm that both are single copy genes.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Amplification, Sequencing

    TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

    doi: 10.1371/journal.pntd.0007256

    Figure Lengend Snippet: TcAUK1 localization in the different forms of T . cruzi . (A) Epimastigote forms at different points of the cell cycle (1F1K1N, 2F2K1N, 2F2K2N) were co-immunostained with rabbit antiserum to TcAUK1 and mouse KMX-1 for TcAUK1 and mitotic spindle, respectively. Yellow arrowhead indicates cells where the nuclei have not segregated yet, and white arrowhead points cells where both nuclei are segregating and (B) Amastigote and Trypomastigote forms isolated from culture supernatants were immunostained with rabbit antiserum to TcAUK1. (C) Infected Vero cells were stained with rhodamine-conjugated phalloidin for actin filaments and intracellular parasites were immunostained with rabbit antiserum to TcAUK1. In all cases, to dye DNA structures–nucleus (n or N) and kinetoplast (k)–cells were counterstained with DAPI. White squares point the magnified regions.

    Article Snippet: When TcAUK1 or TcAUK2 probes were used, the results confirm that both are single copy genes.

    Techniques: Isolation, Infection, Staining

    Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

    doi: 10.1371/journal.pntd.0007256

    Figure Lengend Snippet: Effect of TcAUK1 overexpression on cell cycle progression in epimastigotes. (A) Mitotic progression of pTREX and pTREX-TcAUK1 synchronized cells was monitored by flow cytometry, measuring DNA content every 30 min between hours 11 to 14 post-releasing. (B) Mitotic spindle dynamics and DNA structure duplication (nucleus and kinetoplast) in WT cells were observed by immunostaining with mouse KMX-1 monoclonal antibody and DAPI dye, respectively. In the schematic representation of the events captured by microscopy: green is the mitotic spindle, blue the nucleus and dark blue the kinetoplast; NF means new flagellum and FP is flagellar pocket. (C) Graphical representation of cells with different number of flagellum (F), nucleus (N) and kinetoplast (K) in synchronized control (pTREX) and overexpression cells (pTREX-TcAUK1) at different time points after realizing (11 to 14 hs). Data are presented as the percentage of over 200 cells counted. (D) Mitotic spindle assembling (yellow arrows) and flagellum duplication in TcAUK1 overexpressing cells by immunostaining with mouse monoclonal KMX-1 antibody. Cell’s nucleus and kinetoplast (red arrows) were counterstained with DAPI.

    Article Snippet: When TcAUK1 or TcAUK2 probes were used, the results confirm that both are single copy genes.

    Techniques: Over Expression, Flow Cytometry, Cytometry, Immunostaining, Microscopy

    TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Aurora kinase protein family in Trypanosoma cruzi: Novel role of an AUK-B homologue in kinetoplast replication

    doi: 10.1371/journal.pntd.0007256

    Figure Lengend Snippet: TcAUK1 localization in synchronized overexpressing epimastigotes. TcAUK1 overexpressing epimastigotes, at different phases of cell cycle, were stained for mitotic spindle (mouse monoclonal KMX-1 antibody), TcAUK1 (rabbit antiserum to TcAUK1) and nucleus/kinetoplast (DAPI).

    Article Snippet: When TcAUK1 or TcAUK2 probes were used, the results confirm that both are single copy genes.

    Techniques: Staining