Structured Review

Thermo Fisher sirna
Cellular uptake. Cellular uptake of <t>DP-CLPs–PTX–survivin</t> <t>siRNA</t> (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.
Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sirna/product/Thermo Fisher
Average 99 stars, based on 18683 article reviews
Price from $9.99 to $1999.99
sirna - by Bioz Stars, 2020-08
99/100 stars

Images

1) Product Images from "Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma"

Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

Journal: Drug Delivery

doi: 10.1080/10717544.2018.1494225

Cellular uptake. Cellular uptake of DP-CLPs–PTX–survivin siRNA (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.
Figure Legend Snippet: Cellular uptake. Cellular uptake of DP-CLPs–PTX–survivin siRNA (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.

Techniques Used: Fluorescence, Microscopy, Incubation

Characterization of nanocomplex. (A) Agarose gel electrophoretic mobility shift assay was performed for DP-CLPs–PTX–survivin siRNA (M-e 1 ), DP-CLPs–scrambled siRNA (M-e 2 ), CLPs–PTX–survivin siRNA (M-e 3 ) and CLPs–scrambled siRNA (M-e 4 ). Control was in lane M. Lanes a, b, c, d and e corresponded to lipid/siRNA ratios of 1:0.05, 1:0.08, 1:0.1, 1:0.15 and 1:0.2 (w/w), respectively. (B) Atomic force microscopy pictures of (a) CLPs–scrambled siRNA, (b) DP-CLPs–scrambled siRNA, (c) CLPs–PTX–survivin siRNA siRNA and (d) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08. (C) PTX and DiR in vitro release profiles from the above liposomes. (D) TEM of (a) CLPs–PTX–survivin siRNA siRNA and (b) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08.
Figure Legend Snippet: Characterization of nanocomplex. (A) Agarose gel electrophoretic mobility shift assay was performed for DP-CLPs–PTX–survivin siRNA (M-e 1 ), DP-CLPs–scrambled siRNA (M-e 2 ), CLPs–PTX–survivin siRNA (M-e 3 ) and CLPs–scrambled siRNA (M-e 4 ). Control was in lane M. Lanes a, b, c, d and e corresponded to lipid/siRNA ratios of 1:0.05, 1:0.08, 1:0.1, 1:0.15 and 1:0.2 (w/w), respectively. (B) Atomic force microscopy pictures of (a) CLPs–scrambled siRNA, (b) DP-CLPs–scrambled siRNA, (c) CLPs–PTX–survivin siRNA siRNA and (d) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08. (C) PTX and DiR in vitro release profiles from the above liposomes. (D) TEM of (a) CLPs–PTX–survivin siRNA siRNA and (b) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08.

Techniques Used: Agarose Gel Electrophoresis, Electrophoretic Mobility Shift Assay, Microscopy, In Vitro, Transmission Electron Microscopy

(A) Quantification of the viability of U251-CD133 + cells, U251-CD133 – cells and BCECs after treatment with DMEM, CLPs–scrambled siRNA, DP-CLPs–scrambled siRNA, PTX, CLPs–PTX–survivin siRNA siRNA or DP-CLPs–PTX–survivin siRNA for 48 h. * p
Figure Legend Snippet: (A) Quantification of the viability of U251-CD133 + cells, U251-CD133 – cells and BCECs after treatment with DMEM, CLPs–scrambled siRNA, DP-CLPs–scrambled siRNA, PTX, CLPs–PTX–survivin siRNA siRNA or DP-CLPs–PTX–survivin siRNA for 48 h. * p

Techniques Used:

(A) In vivo fluorescence imaging of intracranial U251-CD133 + glioma tumor-bearing nude mice treated for 24 h with CLPs–PTX–survivin siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ) liposomes, as well as corresponding dissected organs (A 1 and C 1 ). (B) Lesions in nude mice implanted in situ with U251-CD133 + cells after treatment with CLPs–scrambled siRNA (A 1 ), CLPs–PTX–survivin siRNA (C 1 ), DP-CLPs–scrambled siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ); all lesions were characterized by MRI at 19 days post-injection. U251-CD133 + cells were intracranially implanted in nude mice after 48 h treatment with Taxol, CLPs–PTX–survivin siRNA or DP-CLPs–PTX–survivin siRNA. Tumor size was measured 19 days post-injection ( n = 5/group). ** p
Figure Legend Snippet: (A) In vivo fluorescence imaging of intracranial U251-CD133 + glioma tumor-bearing nude mice treated for 24 h with CLPs–PTX–survivin siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ) liposomes, as well as corresponding dissected organs (A 1 and C 1 ). (B) Lesions in nude mice implanted in situ with U251-CD133 + cells after treatment with CLPs–scrambled siRNA (A 1 ), CLPs–PTX–survivin siRNA (C 1 ), DP-CLPs–scrambled siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ); all lesions were characterized by MRI at 19 days post-injection. U251-CD133 + cells were intracranially implanted in nude mice after 48 h treatment with Taxol, CLPs–PTX–survivin siRNA or DP-CLPs–PTX–survivin siRNA. Tumor size was measured 19 days post-injection ( n = 5/group). ** p

Techniques Used: In Vivo, Fluorescence, Imaging, Mouse Assay, In Situ, Magnetic Resonance Imaging, Injection

2) Product Images from "Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP"

Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00304

Knockdown of CacyBP facilitated viral replication and arrested apoptosis in DF-1 cells. DF-1 cells were transfected with mock and/or NC and si-CacyBP/SIP and incubated for 36 h. Mock, treated with transfection regent; NC, transfected with si-NC; si183/326/644, separately transfected with those siRNAs targets CacyBP/SIP. (A) Endogenous CacyBP/SIP was detected by western blotting. (B) DF-1 cells were co-transfected with si-CacyBP (326) and pCMV-HA-CacyBP. Thirty-six hours after transfection, protein expression of HA-CacyBP was detected by anti-HA antibody through immunofluorescence in DF-1 cells. (C) Replication kinetics of NDV RNAs from the mock, NC, and siRNA (326) groups of DF-1 cells; 1 MOI of F48E9 NDV were inoculated into the three groups of cells 24 h after transfection. Q-PCR was used to measure viral RNA replication 24 hpi. (D) Viral plaque formation tests were further used to measure the number of viruses in the supernatants. (E) Three cell cultures were prepared and transfected with mock, NC and siRNA (326), and then, the cells were washed and harvested, an annexin V assay was followed by flow cytometry to monitor for percentage of cells undergoing early apoptosis (bottom right quadrant) and late apoptosis(upper right quadrant). Y-axis is PI signal; X-asis is annexin V-FITC signal. Right graph data were the percentage of total apoptosis (bottom and upper right quadrant)and data from three independent experiments. (F–H) Q-PCR was used to analyze the expression of apoptosis-related markers and immune-associated markers 24 h after transfection with NC and si-CacyBP/SIP. (G) The transfected cell lysate was analyzed by western blotting with the indicated apoptosis-related antibody. Data shown in (C,D) are mean ± SD of four independent experiments, in (E,G,H) are mean ± SD of three independent expriments. * P
Figure Legend Snippet: Knockdown of CacyBP facilitated viral replication and arrested apoptosis in DF-1 cells. DF-1 cells were transfected with mock and/or NC and si-CacyBP/SIP and incubated for 36 h. Mock, treated with transfection regent; NC, transfected with si-NC; si183/326/644, separately transfected with those siRNAs targets CacyBP/SIP. (A) Endogenous CacyBP/SIP was detected by western blotting. (B) DF-1 cells were co-transfected with si-CacyBP (326) and pCMV-HA-CacyBP. Thirty-six hours after transfection, protein expression of HA-CacyBP was detected by anti-HA antibody through immunofluorescence in DF-1 cells. (C) Replication kinetics of NDV RNAs from the mock, NC, and siRNA (326) groups of DF-1 cells; 1 MOI of F48E9 NDV were inoculated into the three groups of cells 24 h after transfection. Q-PCR was used to measure viral RNA replication 24 hpi. (D) Viral plaque formation tests were further used to measure the number of viruses in the supernatants. (E) Three cell cultures were prepared and transfected with mock, NC and siRNA (326), and then, the cells were washed and harvested, an annexin V assay was followed by flow cytometry to monitor for percentage of cells undergoing early apoptosis (bottom right quadrant) and late apoptosis(upper right quadrant). Y-axis is PI signal; X-asis is annexin V-FITC signal. Right graph data were the percentage of total apoptosis (bottom and upper right quadrant)and data from three independent experiments. (F–H) Q-PCR was used to analyze the expression of apoptosis-related markers and immune-associated markers 24 h after transfection with NC and si-CacyBP/SIP. (G) The transfected cell lysate was analyzed by western blotting with the indicated apoptosis-related antibody. Data shown in (C,D) are mean ± SD of four independent experiments, in (E,G,H) are mean ± SD of three independent expriments. * P

Techniques Used: Transfection, Incubation, Western Blot, Expressing, Immunofluorescence, Polymerase Chain Reaction, Annexin V Assay, Flow Cytometry, Cytometry

3) Product Images from "Targeting c-MET by Tivantinib through synergistic activation of JNK/c-jun pathway in cholangiocarcinoma"

Article Title: Targeting c-MET by Tivantinib through synergistic activation of JNK/c-jun pathway in cholangiocarcinoma

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1460-1

The synergistic activation of JNK/c-jun pathway plays a functional role in determining the apoptotic effect of Tivantinib. a Effect on the expression of JNK and c-jun by co-transfection of JNK-specific siRNA oligonucleotide sequences (each at the concentration of 25 and 50 nM) in HuCC-T1 and EGI-1 cell were assessed by western blotting; medium or non-coding siRNA (Ctrl-siRNA) were used as control. b Cell viability after transfection of siRNA targeting JNK in HuCC-T1 and EGI-1 cell. * p
Figure Legend Snippet: The synergistic activation of JNK/c-jun pathway plays a functional role in determining the apoptotic effect of Tivantinib. a Effect on the expression of JNK and c-jun by co-transfection of JNK-specific siRNA oligonucleotide sequences (each at the concentration of 25 and 50 nM) in HuCC-T1 and EGI-1 cell were assessed by western blotting; medium or non-coding siRNA (Ctrl-siRNA) were used as control. b Cell viability after transfection of siRNA targeting JNK in HuCC-T1 and EGI-1 cell. * p

Techniques Used: Activation Assay, Functional Assay, Expressing, Cotransfection, Concentration Assay, Western Blot, Transfection

4) Product Images from "Melatonin receptor depletion suppressed hCG-induced testosterone expression in mouse Leydig cells"

Article Title: Melatonin receptor depletion suppressed hCG-induced testosterone expression in mouse Leydig cells

Journal: Cellular & Molecular Biology Letters

doi: 10.1186/s11658-019-0147-z

siRNA-mediated knockdown of MTNR1A and MTNR1B in mLTC-1 cells. a and b . qPCR assay of the MTNR1A and MTNR1B mRNA levels in mLTC-1 after transfection of siRNAs against MTNR1A and MTNR1B, respectively. c and d . Western blotting results of MTNR1A and MTNR1B protein levels in mLTC-1 after transfection of siRNAs against MTNR1A and MTNR1B, respectively. e and f . The intensity of WB bands in C and D was evaluated with ImageJ. Data are presented as mean ± SEM of triplicate experiments. * P
Figure Legend Snippet: siRNA-mediated knockdown of MTNR1A and MTNR1B in mLTC-1 cells. a and b . qPCR assay of the MTNR1A and MTNR1B mRNA levels in mLTC-1 after transfection of siRNAs against MTNR1A and MTNR1B, respectively. c and d . Western blotting results of MTNR1A and MTNR1B protein levels in mLTC-1 after transfection of siRNAs against MTNR1A and MTNR1B, respectively. e and f . The intensity of WB bands in C and D was evaluated with ImageJ. Data are presented as mean ± SEM of triplicate experiments. * P

Techniques Used: Real-time Polymerase Chain Reaction, Transfection, Western Blot

MTNR1A and MTNR1B regulated steroidogenic gene expression in mLTC-1 cells. a . mLTC-1 cells after transfection of si-Mtnr1a and si-Mtnr1b were treated with 1 IU/ml hCG for 6 h before monitoring of mRNA expression levels of p450scc, P450c17 and StAR using qPCR assay. b . Western blotting results of p450c17 and StAR protein expression in mLTC-1 after knockdown of MTNR1A and MTNR1B using specific siRNA
Figure Legend Snippet: MTNR1A and MTNR1B regulated steroidogenic gene expression in mLTC-1 cells. a . mLTC-1 cells after transfection of si-Mtnr1a and si-Mtnr1b were treated with 1 IU/ml hCG for 6 h before monitoring of mRNA expression levels of p450scc, P450c17 and StAR using qPCR assay. b . Western blotting results of p450c17 and StAR protein expression in mLTC-1 after knockdown of MTNR1A and MTNR1B using specific siRNA

Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot

Effect of MTNR1A and MTNR1B knockdown on testosterone secretion in mLTC-1. a . mLTC-1 cells were treated with 0, 0.5, 1.0 and 5.0 IU/ml of hCG for 6 h before detection of testosterone level using ELISA. The results indicate that 1 IU/ml is the optimal dose for testosterone synthesis. b and c . ELISA results of testosterone secretion after interference of MTNR1A or MTNR1B using siRNA in mLTC-1. d . ELISA result of testosterone secretion after interference with both MTNR1A and MTNR1B in mLTC-1. Data are presented as mean ± SEM of triplicate experiments. * P
Figure Legend Snippet: Effect of MTNR1A and MTNR1B knockdown on testosterone secretion in mLTC-1. a . mLTC-1 cells were treated with 0, 0.5, 1.0 and 5.0 IU/ml of hCG for 6 h before detection of testosterone level using ELISA. The results indicate that 1 IU/ml is the optimal dose for testosterone synthesis. b and c . ELISA results of testosterone secretion after interference of MTNR1A or MTNR1B using siRNA in mLTC-1. d . ELISA result of testosterone secretion after interference with both MTNR1A and MTNR1B in mLTC-1. Data are presented as mean ± SEM of triplicate experiments. * P

Techniques Used: Enzyme-linked Immunosorbent Assay

5) Product Images from "A ribonucleoprotein octamer for targeted siRNA delivery"

Article Title: A ribonucleoprotein octamer for targeted siRNA delivery

Journal: Nature biomedical engineering

doi:

Schematics and characterizations of RNP 8 and its intermediates (a) Assembly between multivalent dsRBD and siRNA-targeting ligand bioconjugates, resulting in cargo loading with precisely controlled stoichiometry and highly accessible targeting ligands. (b) Oligomerization of dsRBD-azide on 8-arm DBCO-PEG via copper-free click chemistry. SDS-PAGE gel electrophoresis indicates that increasing molar ratio of dsRBD over PEG eventually leads to saturated PEGylation (formation of octamer). (c) Separation of dsRBD octamer from excess dsRBD using Superdex™ 200 size exclusion chromatography. (d) Titration of varying amount of dsRBD octamer to siRNA of fixed quantity. 20 pmol of FAM-labeled siRNA is titrated with varying amount of dsRBD octamer (from left to right: 0, 1.25, 2.5, 5, 10, 15, 20 and 40 pmol. RNP 8 is obtained at molar ratio 16/1 and 8/1 (2 nd and 3 rd lanes from the left). More dsRBD octamer leads to unsaturated octamer with smaller gel shifts (4 th -8 th lanes). Additional fine titration studies near ratio 8 can be found in Supplementary Figure S4 . (e) Stability of RNP 8 in 50% mouse serum at 37 °C. Dissociated siRNA is quantified against the pure siRNA band (1 st lane from the left). Quantitative dot plot of the gel band fluorescence intensities is shown in Supplementary Figure S5 . The dissociation is slow with a half-life of approximately 18 h. (f) TEM micrographs of RNP 8 stained by uranyl formate showing uniform and compact nanoparticles. (g) Selected raw images of 7 representative RNP 8 particles. Raw images are shown in the upper row, and the corresponding low-pass filter processed and masked noise-reduced images are shown in the middle row and bottom row, respectively.
Figure Legend Snippet: Schematics and characterizations of RNP 8 and its intermediates (a) Assembly between multivalent dsRBD and siRNA-targeting ligand bioconjugates, resulting in cargo loading with precisely controlled stoichiometry and highly accessible targeting ligands. (b) Oligomerization of dsRBD-azide on 8-arm DBCO-PEG via copper-free click chemistry. SDS-PAGE gel electrophoresis indicates that increasing molar ratio of dsRBD over PEG eventually leads to saturated PEGylation (formation of octamer). (c) Separation of dsRBD octamer from excess dsRBD using Superdex™ 200 size exclusion chromatography. (d) Titration of varying amount of dsRBD octamer to siRNA of fixed quantity. 20 pmol of FAM-labeled siRNA is titrated with varying amount of dsRBD octamer (from left to right: 0, 1.25, 2.5, 5, 10, 15, 20 and 40 pmol. RNP 8 is obtained at molar ratio 16/1 and 8/1 (2 nd and 3 rd lanes from the left). More dsRBD octamer leads to unsaturated octamer with smaller gel shifts (4 th -8 th lanes). Additional fine titration studies near ratio 8 can be found in Supplementary Figure S4 . (e) Stability of RNP 8 in 50% mouse serum at 37 °C. Dissociated siRNA is quantified against the pure siRNA band (1 st lane from the left). Quantitative dot plot of the gel band fluorescence intensities is shown in Supplementary Figure S5 . The dissociation is slow with a half-life of approximately 18 h. (f) TEM micrographs of RNP 8 stained by uranyl formate showing uniform and compact nanoparticles. (g) Selected raw images of 7 representative RNP 8 particles. Raw images are shown in the upper row, and the corresponding low-pass filter processed and masked noise-reduced images are shown in the middle row and bottom row, respectively.

Techniques Used: SDS Page, Nucleic Acid Electrophoresis, Size-exclusion Chromatography, Titration, Labeling, Fluorescence, Transmission Electron Microscopy, Staining

6) Product Images from "Inositol Polyphosphate 4-phosphatase Type II Regulation of Androgen Receptor Activity"

Article Title: Inositol Polyphosphate 4-phosphatase Type II Regulation of Androgen Receptor Activity

Journal: Oncogene

doi: 10.1038/s41388-018-0498-3

Suppression of Akt and PKC signaling contributes to INPP4B regulation of AR transcriptional activity. (A) VCaP cells were transfected with control, INPP4B, or PTEN specific siRNAs. Expression levels of INPP4B, PTEN, pPKCζ, pPKCβII and β-tubulin were measured by Western blotting. (B) LNCaP cells were transfected with control or INPP4B siRNAs for 48 hours in medium supplemented with 10% FBS. Cellular lysates were assayed for INPP4B, pAkt, pPKCζ, pPKCβII and β-tubulin by Western blotting. (C) C4–2 cells were transfected and assayed in parallel with B. (D) LNCaP cells were plated in complete medium and treated with indicated inhibitors for 8 hours. Protein extracts were assayed for AR, INPP4B, pS6, and tubulin levels by Western blotting. (E-J) LNCaP cells were plated in medium supplemented with 10% CSS with either vehicle or 1 nM R1881. Twenty four hours later cells were treated with vehicle (DMSO), 5μM AZD5363, or 2 μM BIM-I for an additional 8 hours. In parallel, LNCaP cells were transfected with control or INPP4B siRNA for 48 hours in 10% CSS medium supplemented with 1 nM R1881. RNA was purified, reverse transcribed, and expression levels of AR regulated genes PLA2G2A (E), KIAA1324 (F), TARP (G), NNMT (H), TMPRSS2 (I) and SYTL2 (J) compared by RT-qPCR. ** represents p
Figure Legend Snippet: Suppression of Akt and PKC signaling contributes to INPP4B regulation of AR transcriptional activity. (A) VCaP cells were transfected with control, INPP4B, or PTEN specific siRNAs. Expression levels of INPP4B, PTEN, pPKCζ, pPKCβII and β-tubulin were measured by Western blotting. (B) LNCaP cells were transfected with control or INPP4B siRNAs for 48 hours in medium supplemented with 10% FBS. Cellular lysates were assayed for INPP4B, pAkt, pPKCζ, pPKCβII and β-tubulin by Western blotting. (C) C4–2 cells were transfected and assayed in parallel with B. (D) LNCaP cells were plated in complete medium and treated with indicated inhibitors for 8 hours. Protein extracts were assayed for AR, INPP4B, pS6, and tubulin levels by Western blotting. (E-J) LNCaP cells were plated in medium supplemented with 10% CSS with either vehicle or 1 nM R1881. Twenty four hours later cells were treated with vehicle (DMSO), 5μM AZD5363, or 2 μM BIM-I for an additional 8 hours. In parallel, LNCaP cells were transfected with control or INPP4B siRNA for 48 hours in 10% CSS medium supplemented with 1 nM R1881. RNA was purified, reverse transcribed, and expression levels of AR regulated genes PLA2G2A (E), KIAA1324 (F), TARP (G), NNMT (H), TMPRSS2 (I) and SYTL2 (J) compared by RT-qPCR. ** represents p

Techniques Used: Activity Assay, Transfection, Expressing, Western Blot, Purification, Quantitative RT-PCR

INPP4B transcription is regulated by full length AR and FOXA1. LNCaP cells in 10% CSS were transfected with noncoding control or FOXA1 specific siRNA; cells were treated with R1881 after 24 hours. Cells were harvested after 48 hours and assayed for FOXA1 expression by western blotting (A), RNA isolated and assayed for FKBP5 (B), RASSF3 (C), and INPP4B (D). ( E) A diagram of AR, FOXA1, and Pol2 recruitment and RNA-seq in LNCaP cells. (F-J) LNCaP AR-V7/pHage cells were placed in medium with 10% CSS for 24 hours and treated with 50 ng/ml Dox or 10 nM R1881 overnight as indicated. Recruitment of AR-FL and AR-V7 to PSA enhancer (F), INPP4B intron 2 (G), INPP4B enhancer region (H), INPP4B transcription start site chr4:143522349 (I), and chr4:143514774 (J) were measured by ChIP-qPCR using Igg as control. * p
Figure Legend Snippet: INPP4B transcription is regulated by full length AR and FOXA1. LNCaP cells in 10% CSS were transfected with noncoding control or FOXA1 specific siRNA; cells were treated with R1881 after 24 hours. Cells were harvested after 48 hours and assayed for FOXA1 expression by western blotting (A), RNA isolated and assayed for FKBP5 (B), RASSF3 (C), and INPP4B (D). ( E) A diagram of AR, FOXA1, and Pol2 recruitment and RNA-seq in LNCaP cells. (F-J) LNCaP AR-V7/pHage cells were placed in medium with 10% CSS for 24 hours and treated with 50 ng/ml Dox or 10 nM R1881 overnight as indicated. Recruitment of AR-FL and AR-V7 to PSA enhancer (F), INPP4B intron 2 (G), INPP4B enhancer region (H), INPP4B transcription start site chr4:143522349 (I), and chr4:143514774 (J) were measured by ChIP-qPCR using Igg as control. * p

Techniques Used: Transfection, Expressing, Western Blot, Isolation, RNA Sequencing Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Reciprocal regulation of AR-V7 and INPP4B. (A) LNCaP AR-V7/pHage cells were placed in medium with 10% CSS or 10% TET FBS serum for 24 hours and treated with 50 ng/ml Dox or 1 nM R1881 as indicated for an additional 36 hours. Protein level of INPP4B, AR-FL, AR-V7, pAkt, pPKCβII, and tubulin were assessed by Western blotting. (B, C) LNCaP AR-V7/pHage cells were transfected with control or INPP4B siRNA and treated with either vehicle or 25 ng/ml Dox for additional 24 hours. Gene expression of INPP4B (B) and EDN2 (C) were analyzed by RT-qPCR using 18S as a control. (D-I) LNCaP AR-V7/pHage cells were transfected and treated as in B. RNA was analyzed for expression of NNMT (D), PSA (E), TARP (F), TMPRSS2 (G), KIAA1324 (H), and SYTL2 (I).*** p
Figure Legend Snippet: Reciprocal regulation of AR-V7 and INPP4B. (A) LNCaP AR-V7/pHage cells were placed in medium with 10% CSS or 10% TET FBS serum for 24 hours and treated with 50 ng/ml Dox or 1 nM R1881 as indicated for an additional 36 hours. Protein level of INPP4B, AR-FL, AR-V7, pAkt, pPKCβII, and tubulin were assessed by Western blotting. (B, C) LNCaP AR-V7/pHage cells were transfected with control or INPP4B siRNA and treated with either vehicle or 25 ng/ml Dox for additional 24 hours. Gene expression of INPP4B (B) and EDN2 (C) were analyzed by RT-qPCR using 18S as a control. (D-I) LNCaP AR-V7/pHage cells were transfected and treated as in B. RNA was analyzed for expression of NNMT (D), PSA (E), TARP (F), TMPRSS2 (G), KIAA1324 (H), and SYTL2 (I).*** p

Techniques Used: Western Blot, Transfection, Expressing, Quantitative RT-PCR

7) Product Images from "ERBB2-modulated ATG4B and autophagic cell death in human ARPE19 during oxidative stress"

Article Title: ERBB2-modulated ATG4B and autophagic cell death in human ARPE19 during oxidative stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0213932

Kinome siRNA screening for cytotoxic effects of ARPE-19 cells during oxidative stress. (A) Human RPE ARPE-19 cells were treated with non-targeting siRNA for 48 h, followed by treatment with hydrogen peroxide at 62.5, 125, 250, 500, and 1000 μM for 24 h in order to determine cell viability. Cellular ROS production and (B) cell viability were measured with ROS-Glo and Cell-titer Glo, respectively. (C) Cells were treated with kinome siRNA (710 gene) for 48 h followed by treatment with hydrogen peroxide (500 μM) for 24 h in order to measure ROS production in cells. (D) The top 10-ranked hits from kinome siRNA screening were further validated for cellular ROS production in three independent experiments (Three parallel samples were included in each experiment), and the results are shown as mean ± SEM.
Figure Legend Snippet: Kinome siRNA screening for cytotoxic effects of ARPE-19 cells during oxidative stress. (A) Human RPE ARPE-19 cells were treated with non-targeting siRNA for 48 h, followed by treatment with hydrogen peroxide at 62.5, 125, 250, 500, and 1000 μM for 24 h in order to determine cell viability. Cellular ROS production and (B) cell viability were measured with ROS-Glo and Cell-titer Glo, respectively. (C) Cells were treated with kinome siRNA (710 gene) for 48 h followed by treatment with hydrogen peroxide (500 μM) for 24 h in order to measure ROS production in cells. (D) The top 10-ranked hits from kinome siRNA screening were further validated for cellular ROS production in three independent experiments (Three parallel samples were included in each experiment), and the results are shown as mean ± SEM.

Techniques Used:

Effects of ERBB2 on autophagy in ARPE-19 cells during oxidative stress. (A) Human RPE ARPE-19 cells were transfected with 5 nM scramble siRNA or siRNA against ERBB2 for 64 h and then treated with hydrogen peroxide (500 μM) for 8 hr. The cells were lysed for western blotting using antibodies against NRF2, SQSTM1, MAP1LC3B, ATG4B, and ACTB. (B) The quantitative results for ratio of MAP1LC3B-II/I and SQSTM1 protein level are shown. (C) The mRNA levels of SQSTM1 in cells as mentioned above were determined by real-time polymerase chain reaction (PCR). The results were analyzed using Prism 5.0 and expressed as mean ± SEM from three independent experiments (Three parallel samples were included in each experiment).
Figure Legend Snippet: Effects of ERBB2 on autophagy in ARPE-19 cells during oxidative stress. (A) Human RPE ARPE-19 cells were transfected with 5 nM scramble siRNA or siRNA against ERBB2 for 64 h and then treated with hydrogen peroxide (500 μM) for 8 hr. The cells were lysed for western blotting using antibodies against NRF2, SQSTM1, MAP1LC3B, ATG4B, and ACTB. (B) The quantitative results for ratio of MAP1LC3B-II/I and SQSTM1 protein level are shown. (C) The mRNA levels of SQSTM1 in cells as mentioned above were determined by real-time polymerase chain reaction (PCR). The results were analyzed using Prism 5.0 and expressed as mean ± SEM from three independent experiments (Three parallel samples were included in each experiment).

Techniques Used: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Effects of autophagy inhibitors in oxidative stress-induced cell death. (A) Human RPE ARPE-19 cells were treated with hydrogen peroxide (500 μM) in the absence or presence of autophagy inhibitor CQ (20 μM) or ConA (10 nM) for 24 h. Cell viability was quantified with Cell-titer Glo assay system. (B) The cells were transfected with 5 nM scramble siRNA or siRNA against ERBB2 for 48 h and treated with hydrogen peroxide (500 μM) in the absence or presence of autophagy inhibitors CQ (20 μM) or ConA (10 nM) for 24 h. The results were analyzed with Prism 5 and expressed as mean ± SEM from three independent experiments (Three parallel samples were included in each experiment).
Figure Legend Snippet: Effects of autophagy inhibitors in oxidative stress-induced cell death. (A) Human RPE ARPE-19 cells were treated with hydrogen peroxide (500 μM) in the absence or presence of autophagy inhibitor CQ (20 μM) or ConA (10 nM) for 24 h. Cell viability was quantified with Cell-titer Glo assay system. (B) The cells were transfected with 5 nM scramble siRNA or siRNA against ERBB2 for 48 h and treated with hydrogen peroxide (500 μM) in the absence or presence of autophagy inhibitors CQ (20 μM) or ConA (10 nM) for 24 h. The results were analyzed with Prism 5 and expressed as mean ± SEM from three independent experiments (Three parallel samples were included in each experiment).

Techniques Used: Glo Assay, Transfection

Effects of ERBB2 on ATG4B in ARPE-19 cell during oxidative stress. Cells were transfected with 5 nM scramble siRNA or siRNA against ERBB2 or ATG4B for 48 h, followed by treatment with hydrogen peroxide (500 μM) for 24 h. The cells were then lysed, and equal amount of proteins were incubated with S-tagged (A) MAP1LC3B and (C) GATE-16 for 2 h. S-tag removal and ATG4B expression were examined by immunoblotting (B and D). The S-tag and ATG4B protein levels were quantitated with image J and expressed as mean ± SEM. (E) The knock-downed cells in the absence or presence of hydrogen peroxide were harvested, and nuclear and cytoplasmic fractions were split. The fractionated proteins were determined by immunoblotting using antibodies against NRF2 and ATG4B. (F) NRF2 transcriptional activity was monitored in cells harboring vector containing NRF2 promoter and luciferase.
Figure Legend Snippet: Effects of ERBB2 on ATG4B in ARPE-19 cell during oxidative stress. Cells were transfected with 5 nM scramble siRNA or siRNA against ERBB2 or ATG4B for 48 h, followed by treatment with hydrogen peroxide (500 μM) for 24 h. The cells were then lysed, and equal amount of proteins were incubated with S-tagged (A) MAP1LC3B and (C) GATE-16 for 2 h. S-tag removal and ATG4B expression were examined by immunoblotting (B and D). The S-tag and ATG4B protein levels were quantitated with image J and expressed as mean ± SEM. (E) The knock-downed cells in the absence or presence of hydrogen peroxide were harvested, and nuclear and cytoplasmic fractions were split. The fractionated proteins were determined by immunoblotting using antibodies against NRF2 and ATG4B. (F) NRF2 transcriptional activity was monitored in cells harboring vector containing NRF2 promoter and luciferase.

Techniques Used: Transfection, Incubation, Expressing, Activity Assay, Plasmid Preparation, Luciferase

Effects of ERBB2 in autophagy deficient ARPE-19 cells during oxidative stress. (A) Human RPE ARPE-19 cells were transfected with 5 nM scramble siRNA or siRNA against ERBB2 without or with ULK1, BECN1, ATG5, and ATG7 for 48 h and treated with hydrogen peroxide (500 μM) for 24 h. The cells were lysed for immunoblotting to determine protein level of ERBB2, ATG5, ATG7, BECN1, ULK1, SQSTM1, and MAP1LC3B using ACTB as the internal control. (B) SQSTM1 protein levels and ratio of MAP1LC3B-II/I were quantitated with image J and expressed as mean ± SEM. (C) The knock-downed cells were treated with hydrogen peroxide (500 μM) for 8 h, and cell viability was quantified with Celltiter-Glo assay system. (D) Schematic diagram for the potential role of ERBB2 in autophagic cell death in ARPE-19 cells during oxidative stress.
Figure Legend Snippet: Effects of ERBB2 in autophagy deficient ARPE-19 cells during oxidative stress. (A) Human RPE ARPE-19 cells were transfected with 5 nM scramble siRNA or siRNA against ERBB2 without or with ULK1, BECN1, ATG5, and ATG7 for 48 h and treated with hydrogen peroxide (500 μM) for 24 h. The cells were lysed for immunoblotting to determine protein level of ERBB2, ATG5, ATG7, BECN1, ULK1, SQSTM1, and MAP1LC3B using ACTB as the internal control. (B) SQSTM1 protein levels and ratio of MAP1LC3B-II/I were quantitated with image J and expressed as mean ± SEM. (C) The knock-downed cells were treated with hydrogen peroxide (500 μM) for 8 h, and cell viability was quantified with Celltiter-Glo assay system. (D) Schematic diagram for the potential role of ERBB2 in autophagic cell death in ARPE-19 cells during oxidative stress.

Techniques Used: Transfection, Glo Assay

8) Product Images from "Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism"

Article Title: Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism

Journal: Thyroid

doi: 10.1089/thy.2012.0457

Uptake of lipid-conjugated ApoE3 by T3 in HepG2 cells. HepG2 cells incubated in CSS for 24 hours were treated with the indicated concentrations of T3 for 48 hours. Human recombinant ApoE3 conjugated with lipid was added to the culture medium and cells were incubated for 1 hour. (A) Western blot analysis of ApoE3 in HepG2 cells incubated with or without added ApoE3. (B) Western blot analysis of ApoE3 in HepG2 cells transfected with nontargeting negative siRNA (siCTRL) and siRNA targeting LRP1 (siLRP1). (C) Western blot analysis of ApoE3 in HepG2 cells transfected with siCTRL and siRNA targeting the LDL receptor (siLDLR). The human recombinant receptor–associated protein was used as a functional blocker of LRP1 and LDLR. The human recombinant receptor–associated protein was added to the culture medium before adding ApoE3. Three independent experiments were performed for the representative figures. siRNA, small interfering RNA; LDL, low-density lipoprotein; LDLR, LDL receptor.
Figure Legend Snippet: Uptake of lipid-conjugated ApoE3 by T3 in HepG2 cells. HepG2 cells incubated in CSS for 24 hours were treated with the indicated concentrations of T3 for 48 hours. Human recombinant ApoE3 conjugated with lipid was added to the culture medium and cells were incubated for 1 hour. (A) Western blot analysis of ApoE3 in HepG2 cells incubated with or without added ApoE3. (B) Western blot analysis of ApoE3 in HepG2 cells transfected with nontargeting negative siRNA (siCTRL) and siRNA targeting LRP1 (siLRP1). (C) Western blot analysis of ApoE3 in HepG2 cells transfected with siCTRL and siRNA targeting the LDL receptor (siLDLR). The human recombinant receptor–associated protein was used as a functional blocker of LRP1 and LDLR. The human recombinant receptor–associated protein was added to the culture medium before adding ApoE3. Three independent experiments were performed for the representative figures. siRNA, small interfering RNA; LDL, low-density lipoprotein; LDLR, LDL receptor.

Techniques Used: Incubation, Recombinant, Western Blot, Transfection, Functional Assay, Small Interfering RNA

9) Product Images from "Inhibition of Chemokine (CXC Motif) Ligand 12/Chemokine (CXC Motif) Receptor 4 Axis (CXCL12/CXCR4)-mediated Cell Migration by Targeting Mammalian Target of Rapamycin (mTOR) Pathway in Human Gastric Carcinoma Cells *"

Article Title: Inhibition of Chemokine (CXC Motif) Ligand 12/Chemokine (CXC Motif) Receptor 4 Axis (CXCL12/CXCR4)-mediated Cell Migration by Targeting Mammalian Target of Rapamycin (mTOR) Pathway in Human Gastric Carcinoma Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.302299

CXCL12/CXCR4 axis interacted with p110β catalytic subunits and activated PI3K/mTOR signaling pathway. A , down-regulation of p110 subunit by transient transfection of siRNA targeting p110β but not p110α blocked phosphorylation Akt and p70S6K induced by CXCL12. After transfected with respective siRNA for 48 h, MKN-45 cells were treated with CXCL12 (100 ng/ml) for 5 min. Phosphorylated Akt and p70S6K were analyzed by immunoblotting; GAPDH was employed a loading control. B , p110β-specific inhibitor TGX-221 abrogated CXCL12-triggered PI3K/mTOR signaling. Serum-deprived MKN-45 cells were pretreated with TGX-221 for 1 h and then stimulated with CXCL12 for 5 min. C , CXCL12 enhanced the association between p110β and CXCR4. Serum-deprived MKN-45 cells were treated with vehicle or CXCL12 for 5 min. For immunoprecipitation, the primary antibody (1 μg) against p110β was added to 200 μl of cell lysates (2 × 10 6 cells) and incubated with rotation at 4 °C for 2 h. Rabbit IgG was used as a negative control. After precipitation with agarose A/G, proteins were resolved in 100 μl 1×SDS loading buffer, and 20 μl of each sample was separated with 12% SDS-PAGE and analyzed by immunoblotting using the antibody against CXCR4. Protein samples from whole cell lysate were used to indicate the position of the protein examined. D , involvement of G i protein in CXCL12-induced phosphorylation of AKT. Serum-deprived MKN-45 cells were pretreated with pertussis toxin (100 ng/ml) for 2 h and then stimulated with CXCL12 for 5 min. Data shown are representative of at least three independent experiments.
Figure Legend Snippet: CXCL12/CXCR4 axis interacted with p110β catalytic subunits and activated PI3K/mTOR signaling pathway. A , down-regulation of p110 subunit by transient transfection of siRNA targeting p110β but not p110α blocked phosphorylation Akt and p70S6K induced by CXCL12. After transfected with respective siRNA for 48 h, MKN-45 cells were treated with CXCL12 (100 ng/ml) for 5 min. Phosphorylated Akt and p70S6K were analyzed by immunoblotting; GAPDH was employed a loading control. B , p110β-specific inhibitor TGX-221 abrogated CXCL12-triggered PI3K/mTOR signaling. Serum-deprived MKN-45 cells were pretreated with TGX-221 for 1 h and then stimulated with CXCL12 for 5 min. C , CXCL12 enhanced the association between p110β and CXCR4. Serum-deprived MKN-45 cells were treated with vehicle or CXCL12 for 5 min. For immunoprecipitation, the primary antibody (1 μg) against p110β was added to 200 μl of cell lysates (2 × 10 6 cells) and incubated with rotation at 4 °C for 2 h. Rabbit IgG was used as a negative control. After precipitation with agarose A/G, proteins were resolved in 100 μl 1×SDS loading buffer, and 20 μl of each sample was separated with 12% SDS-PAGE and analyzed by immunoblotting using the antibody against CXCR4. Protein samples from whole cell lysate were used to indicate the position of the protein examined. D , involvement of G i protein in CXCL12-induced phosphorylation of AKT. Serum-deprived MKN-45 cells were pretreated with pertussis toxin (100 ng/ml) for 2 h and then stimulated with CXCL12 for 5 min. Data shown are representative of at least three independent experiments.

Techniques Used: Transfection, Immunoprecipitation, Incubation, Negative Control, SDS Page

10) Product Images from "Application of Phi29 Motor pRNA for Targeted Therapeutic Delivery of siRNA Silencing Metallothionein-IIA and Survivin in Ovarian Cancers"

Article Title: Application of Phi29 Motor pRNA for Targeted Therapeutic Delivery of siRNA Silencing Metallothionein-IIA and Survivin in Ovarian Cancers

Journal: Molecular Therapy

doi: 10.1038/mt.2010.243

The RNAi function of pRNA/siRNA is more potent than siRNA. MT-IIA or survivin expression inhibits cell proliferation in OVCA 433 cells. Results are presented as average ± SEM of triplicate experiments. Asterisk indicates t -test P
Figure Legend Snippet: The RNAi function of pRNA/siRNA is more potent than siRNA. MT-IIA or survivin expression inhibits cell proliferation in OVCA 433 cells. Results are presented as average ± SEM of triplicate experiments. Asterisk indicates t -test P

Techniques Used: Expressing

Ba′pRNA/si(CONT2) is localized to GW/P-bodies. ( a ) The Ba′pRNA/si(CONT2) was labeled using the Cy3 Silencer siRNA labeling kit (Ambion, Austin, TX). The resultant Cy3-pRNA/si(CONT2) was analyzed on a 8% denaturing gel. ( b ) OVCA 433 cells were transfected with Cy3-pRNA/si(CONT2) (1–3) and FITC-siRNA (4–6). Cells were examined after 24 hours using an inverted fluorescent microscope. Scale bar represents 20 µmol/l. Panels 3, 6 represent enlargements of the boxed areas ( c ) The Cy3-pRNA/si(CONT2)- (1–6) transfected cells were fixed and immunostained with mouse anti-GW182 antibody, followed by Alexa Fluor 488 chicken anti-mouse secondary antibody. Arrows point to nonoverlapping signals. The closed and open arrows in panel 4 is further magnified in panels 5 and 6. Scale bar represents 5 µmol/l.
Figure Legend Snippet: Ba′pRNA/si(CONT2) is localized to GW/P-bodies. ( a ) The Ba′pRNA/si(CONT2) was labeled using the Cy3 Silencer siRNA labeling kit (Ambion, Austin, TX). The resultant Cy3-pRNA/si(CONT2) was analyzed on a 8% denaturing gel. ( b ) OVCA 433 cells were transfected with Cy3-pRNA/si(CONT2) (1–3) and FITC-siRNA (4–6). Cells were examined after 24 hours using an inverted fluorescent microscope. Scale bar represents 20 µmol/l. Panels 3, 6 represent enlargements of the boxed areas ( c ) The Cy3-pRNA/si(CONT2)- (1–6) transfected cells were fixed and immunostained with mouse anti-GW182 antibody, followed by Alexa Fluor 488 chicken anti-mouse secondary antibody. Arrows point to nonoverlapping signals. The closed and open arrows in panel 4 is further magnified in panels 5 and 6. Scale bar represents 5 µmol/l.

Techniques Used: Labeling, Transfection, Microscopy

Ovarian cell lines express high levels of MT-IIA and survivin mRNA, and their silencing. Total RNA was extracted from OVCA 432, OVCA433, and SKOV-3 ovarian cell lines transfected with the pRNA/siRNA indicated after 24 hours, was reverse transcribed to cDNA, and was analyzed for mRNA expression by qRT-PCR. Values were normalized to GAPDH levels in the respective cell line. Error bars represent the SEM of three experiments, each performed in triplicate. Asterisk indicates statistically significant ( P
Figure Legend Snippet: Ovarian cell lines express high levels of MT-IIA and survivin mRNA, and their silencing. Total RNA was extracted from OVCA 432, OVCA433, and SKOV-3 ovarian cell lines transfected with the pRNA/siRNA indicated after 24 hours, was reverse transcribed to cDNA, and was analyzed for mRNA expression by qRT-PCR. Values were normalized to GAPDH levels in the respective cell line. Error bars represent the SEM of three experiments, each performed in triplicate. Asterisk indicates statistically significant ( P

Techniques Used: Transfection, Expressing, Quantitative RT-PCR

Sequence and structure of pRNA chimera. ( a ) Sketch of chimeric siRNA harbored in pRNA vector. ( a ) Sequence and secondary structure of (1) Ba′pRNA, (2) Ab′pRNA/si(CONT2) and Ba′pRNA/si(CONT2) dimer, (3) Ba′pRNA/si(CONT2) (control siRNA), (4) Ba′pRNA/si(MT2-1), (5) Ba′pRNA/si(MT2-2), and (6) Ba′pRNA/si(Surv). All chimeras have identical pRNA backbone sequence, whereas the sequence of siRNA are altered. ( b ) The pRNA/siRNA have the correct size. The Ab′pRNA, Ba′pRNA/si(MT2-1), Ba′pRNA/si(MT2-2), Ba′pRNA/si(Surv) and Ba′pRNA/si(CONT2) and Ba′pRNA (lanes 1–6) were transcribed and analyzed on a 6% denaturing gel. ( c ) The pRNA/siRNA have the correct intermolecular interactions. The Ba′pRNA, Ba′pRNA/si(MT2-1), Ba′pRNA/si(MT2-2), Ba′pRNA/si(Surv), and Ba′pRNA/si(CONT2) (lanes 1–5, 8–12) were analyzed on a 6% nondenaturing gel in the absence (monomers, lanes 1–5) and presence (dimers, lanes 8–12) of Ab′pRNA. The migration of the monomer Ab′pRNA (lanes 6, 7) and Ba′pRNA (lane 13) is also shown. ( d ) Processing of chimeric pRNA/siRNA complex into siRNA by purified dicer. The Ba′pRNA, Ba′pRNA/si(MT2-1), Ba′pRNA/si(MT2-2), Ba′pRNA/si(Surv), and Ba′pRNA/si(CONT2) (lanes 1–5, 6–10) were incubated in the absence (lanes 1–5) or presence (lanes 6–10) of dicer for 1 hour, and analyzed by 8% denaturing gel. Lane 11 represents Ba′pRNA/si(CONT2) incubated with dicer for 3 hours.
Figure Legend Snippet: Sequence and structure of pRNA chimera. ( a ) Sketch of chimeric siRNA harbored in pRNA vector. ( a ) Sequence and secondary structure of (1) Ba′pRNA, (2) Ab′pRNA/si(CONT2) and Ba′pRNA/si(CONT2) dimer, (3) Ba′pRNA/si(CONT2) (control siRNA), (4) Ba′pRNA/si(MT2-1), (5) Ba′pRNA/si(MT2-2), and (6) Ba′pRNA/si(Surv). All chimeras have identical pRNA backbone sequence, whereas the sequence of siRNA are altered. ( b ) The pRNA/siRNA have the correct size. The Ab′pRNA, Ba′pRNA/si(MT2-1), Ba′pRNA/si(MT2-2), Ba′pRNA/si(Surv) and Ba′pRNA/si(CONT2) and Ba′pRNA (lanes 1–6) were transcribed and analyzed on a 6% denaturing gel. ( c ) The pRNA/siRNA have the correct intermolecular interactions. The Ba′pRNA, Ba′pRNA/si(MT2-1), Ba′pRNA/si(MT2-2), Ba′pRNA/si(Surv), and Ba′pRNA/si(CONT2) (lanes 1–5, 8–12) were analyzed on a 6% nondenaturing gel in the absence (monomers, lanes 1–5) and presence (dimers, lanes 8–12) of Ab′pRNA. The migration of the monomer Ab′pRNA (lanes 6, 7) and Ba′pRNA (lane 13) is also shown. ( d ) Processing of chimeric pRNA/siRNA complex into siRNA by purified dicer. The Ba′pRNA, Ba′pRNA/si(MT2-1), Ba′pRNA/si(MT2-2), Ba′pRNA/si(Surv), and Ba′pRNA/si(CONT2) (lanes 1–5, 6–10) were incubated in the absence (lanes 1–5) or presence (lanes 6–10) of dicer for 1 hour, and analyzed by 8% denaturing gel. Lane 11 represents Ba′pRNA/si(CONT2) incubated with dicer for 3 hours.

Techniques Used: Sequencing, Plasmid Preparation, Migration, Purification, Incubation

Targeted delivery of folate-tagged pRNA into ovarian cancer cells. ( a ) SKOV-3 cells express high levels of folate receptor-α mRNA. Total RNA was extracted from HOSE 642, OVCA 432, OVCA433, and SKOV-3 cell lines, was reverse transcribed to cDNA, and was analyzed for mRNA expression by qRT-PCR. Values were normalized to GAPDH levels in the respective cell line. Error bars represent the SEM of three samples each, each performed in triplicate. Expression levels of survivin are shown relative to HOSE 642 cells. ( b ) The Ba′pRNA/siRNA can dimerize with folate-Ab′pRNA. The Ba′pRNA/siRNA was incubated with folate-Ab′pRNA and analyzed on a nondenaturing gel. The 22/98 Sph I Ab′pRNA (lanes 1, 10), Ab′pRNA (lanes 2, 11), Ba′pRNA (lanes 3, 9, 12, 18), folate-Ab′pRNA (lanes 4, 13), Ba′pRNA/si(MT2-1) (lanes 5, 7), Ba′pRNA/si(MT2-2) (lanes 6, 8), Ba′pRNA/si(Surv) (lanes 14, 16), and Ba′pRNA/si(CONT2) (lanes 15, 17) were analyzed on a 6% nondenaturing gel in the absence (monomers, lanes 1–6, 10–15) and presence (dimers, lanes 7–9, 16–18) of folate-Ab′pRNA. ( c ) The folate-pRNA-Cy3-pRNA/siRNA binds to KB cells. Cells maintained in folate-free media were treated with the indicated Cy3-pRNA/siRNA dimer with either folate-pRNA or NH 2 -pRNA. Flow cytometric analysis was performed to determine the percent of cells bound by Cy3-pRNA/siRNA. ( d ) Effects of targeted silencing MT-IIA or s urvivin expression in SKOV-3 cells. SKOV-3 cells were treated with the dimers formed above. Cell proliferation assays were performed 72 hours post-pRNA/siRNA treatment as indicated.
Figure Legend Snippet: Targeted delivery of folate-tagged pRNA into ovarian cancer cells. ( a ) SKOV-3 cells express high levels of folate receptor-α mRNA. Total RNA was extracted from HOSE 642, OVCA 432, OVCA433, and SKOV-3 cell lines, was reverse transcribed to cDNA, and was analyzed for mRNA expression by qRT-PCR. Values were normalized to GAPDH levels in the respective cell line. Error bars represent the SEM of three samples each, each performed in triplicate. Expression levels of survivin are shown relative to HOSE 642 cells. ( b ) The Ba′pRNA/siRNA can dimerize with folate-Ab′pRNA. The Ba′pRNA/siRNA was incubated with folate-Ab′pRNA and analyzed on a nondenaturing gel. The 22/98 Sph I Ab′pRNA (lanes 1, 10), Ab′pRNA (lanes 2, 11), Ba′pRNA (lanes 3, 9, 12, 18), folate-Ab′pRNA (lanes 4, 13), Ba′pRNA/si(MT2-1) (lanes 5, 7), Ba′pRNA/si(MT2-2) (lanes 6, 8), Ba′pRNA/si(Surv) (lanes 14, 16), and Ba′pRNA/si(CONT2) (lanes 15, 17) were analyzed on a 6% nondenaturing gel in the absence (monomers, lanes 1–6, 10–15) and presence (dimers, lanes 7–9, 16–18) of folate-Ab′pRNA. ( c ) The folate-pRNA-Cy3-pRNA/siRNA binds to KB cells. Cells maintained in folate-free media were treated with the indicated Cy3-pRNA/siRNA dimer with either folate-pRNA or NH 2 -pRNA. Flow cytometric analysis was performed to determine the percent of cells bound by Cy3-pRNA/siRNA. ( d ) Effects of targeted silencing MT-IIA or s urvivin expression in SKOV-3 cells. SKOV-3 cells were treated with the dimers formed above. Cell proliferation assays were performed 72 hours post-pRNA/siRNA treatment as indicated.

Techniques Used: Expressing, Quantitative RT-PCR, Incubation, Flow Cytometry

11) Product Images from "Shared molecular mechanisms regulate multiple catenin proteins: canonical Wnt signals and components modulate p120-catenin isoform-1 and additional p120 subfamily members"

Article Title: Shared molecular mechanisms regulate multiple catenin proteins: canonical Wnt signals and components modulate p120-catenin isoform-1 and additional p120 subfamily members

Journal: Journal of Cell Science

doi: 10.1242/jcs.067199

Upstream Wnt pathway components modulate the levels of p120-catenin. ( A ) Co-injection of Frodo or Wnt8 counter the effectiveness of Myc–GSK3β (5 pg) or Myc–axin (0.1 ng) to reduce HA–p120 (0.5 ng) levels. Embryos expressing up to two exogenous constructs (as noted) in addition to HA–p120 were harvested at gastrulation, and the corresponding lysates immunoblotted (IB) with antibody against HA to detect p120. Actin was used as an internal loading control. Total mRNA injection loads were equalized using β-galactosidase. ( B ) Exogenous Wnts (0.5 ng of Wnt8, Wnt11 or Wnt5a mRNA) were co-injected with both HA–GSK3β (0.1 ng) and HA–p120 (0.25 ng) into both blastomere of two-cell embryos. At stage 12, Wnt protection (versus no protection) from GSK3β impact upon HA–p120 was assessed by immunoblotting. Actin served as an internal loading control. ( C ) An increasing dose of Wnt8 mRNA was co-injected with both HA–p120 (0.5 ng) and Myc–axin (0.5 ng) into two blastomeres of two-cell embryos that were later collected at gastrulation (stage 11, with 20 embryos per condition). The protein levels of p120-catenin were assessed by immunoblotting. ( D ) MDA-231, HeLa and 293T cells were transfected with siRNAs directed against axin-1 and axin-2 (50 pmol), as indicated, for 48 hours. Endogenous p120-catenin levels were monitored through anti-p120 immunoblotting (6H11). Each experiment was repeated at least three times. nc, negative control. ( E ) HeLa cells were transiently transfected with siRNA for LRP5 or LRP6 (50 pmol), along with either Myc-tagged LRP5 or LRP6, and effects assessed by means of Myc-immunoblotting. ( F ) MDA-435 cells were seeded in six-well plates, followed by transfection with LRP5 and LRP6 siRNAs. The effect of LRP5 and LRP6 depletion on p120-catenin was monitored using distinct antibodies directed against p120 (pp120 or 6H11). The asterisk (*) indicates a nonspecific band serving as an additional negative control. ( G ) The stability of p120-catenin in HeLa cells was resolved as described for F. ( H ) Myc–p120 full-length (isoform-1) or ΔN-p120 were transiently transfected into 293T cells for 24 hours, followed by MG132 or D4476 treatment (6 hours) at the doses indicated. The levels of p120 were monitored through Myc-immunoblotting. ( I ) 293T cells were transiently transfected with mouse p120 isoform-1A or isoform-3A. p120-catenin isoforms were monitored in the presence of BIO or D4476 (6 hours), employing an antibody against Myc.
Figure Legend Snippet: Upstream Wnt pathway components modulate the levels of p120-catenin. ( A ) Co-injection of Frodo or Wnt8 counter the effectiveness of Myc–GSK3β (5 pg) or Myc–axin (0.1 ng) to reduce HA–p120 (0.5 ng) levels. Embryos expressing up to two exogenous constructs (as noted) in addition to HA–p120 were harvested at gastrulation, and the corresponding lysates immunoblotted (IB) with antibody against HA to detect p120. Actin was used as an internal loading control. Total mRNA injection loads were equalized using β-galactosidase. ( B ) Exogenous Wnts (0.5 ng of Wnt8, Wnt11 or Wnt5a mRNA) were co-injected with both HA–GSK3β (0.1 ng) and HA–p120 (0.25 ng) into both blastomere of two-cell embryos. At stage 12, Wnt protection (versus no protection) from GSK3β impact upon HA–p120 was assessed by immunoblotting. Actin served as an internal loading control. ( C ) An increasing dose of Wnt8 mRNA was co-injected with both HA–p120 (0.5 ng) and Myc–axin (0.5 ng) into two blastomeres of two-cell embryos that were later collected at gastrulation (stage 11, with 20 embryos per condition). The protein levels of p120-catenin were assessed by immunoblotting. ( D ) MDA-231, HeLa and 293T cells were transfected with siRNAs directed against axin-1 and axin-2 (50 pmol), as indicated, for 48 hours. Endogenous p120-catenin levels were monitored through anti-p120 immunoblotting (6H11). Each experiment was repeated at least three times. nc, negative control. ( E ) HeLa cells were transiently transfected with siRNA for LRP5 or LRP6 (50 pmol), along with either Myc-tagged LRP5 or LRP6, and effects assessed by means of Myc-immunoblotting. ( F ) MDA-435 cells were seeded in six-well plates, followed by transfection with LRP5 and LRP6 siRNAs. The effect of LRP5 and LRP6 depletion on p120-catenin was monitored using distinct antibodies directed against p120 (pp120 or 6H11). The asterisk (*) indicates a nonspecific band serving as an additional negative control. ( G ) The stability of p120-catenin in HeLa cells was resolved as described for F. ( H ) Myc–p120 full-length (isoform-1) or ΔN-p120 were transiently transfected into 293T cells for 24 hours, followed by MG132 or D4476 treatment (6 hours) at the doses indicated. The levels of p120 were monitored through Myc-immunoblotting. ( I ) 293T cells were transiently transfected with mouse p120 isoform-1A or isoform-3A. p120-catenin isoforms were monitored in the presence of BIO or D4476 (6 hours), employing an antibody against Myc.

Techniques Used: Injection, Expressing, Construct, Multiple Displacement Amplification, Transfection, Negative Control

12) Product Images from "Butyrophilin 3A1 Plays an Essential Role in Prenyl Pyrophosphate Stimulation of Human Vγ2Vδ2 T Cells"

Article Title: Butyrophilin 3A1 Plays an Essential Role in Prenyl Pyrophosphate Stimulation of Human Vγ2Vδ2 T Cells

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1300658

siRNA inhibition of BTN3A1 in APC abolishes IPP stimulation of Vγ2Vδ2 T cells. ( A ) siRNA inhibition of BTN3A1, BTN3A2, and BTN3A3 expression. HeLa cells were transfected with either control siRNA or with siRNAs specific for each of the members of the BTN3 family. After 72 h, transfected HeLa cells were stained with the 20.1 mAb and BTN3 surface expression determined by flow cytometry. ( B ) siRNA inhibition of BTN3A1 greatly reduces IPP stimulation of Vγ2Vδ2 T cells. siRNA transfected HeLa cells were used as APC for 20.1 mAb, IPP, and PHA stimulation of the 12G12 Vγ2Vδ2 T cell clone. Note that for each of the siRNA shown, there was at least one additional siRNA with a similar effect (i.e. oligo A for BTN3A1, oligo C for BTN3A2, and oligo A and C for BTN3A3).
Figure Legend Snippet: siRNA inhibition of BTN3A1 in APC abolishes IPP stimulation of Vγ2Vδ2 T cells. ( A ) siRNA inhibition of BTN3A1, BTN3A2, and BTN3A3 expression. HeLa cells were transfected with either control siRNA or with siRNAs specific for each of the members of the BTN3 family. After 72 h, transfected HeLa cells were stained with the 20.1 mAb and BTN3 surface expression determined by flow cytometry. ( B ) siRNA inhibition of BTN3A1 greatly reduces IPP stimulation of Vγ2Vδ2 T cells. siRNA transfected HeLa cells were used as APC for 20.1 mAb, IPP, and PHA stimulation of the 12G12 Vγ2Vδ2 T cell clone. Note that for each of the siRNA shown, there was at least one additional siRNA with a similar effect (i.e. oligo A for BTN3A1, oligo C for BTN3A2, and oligo A and C for BTN3A3).

Techniques Used: Inhibition, Expressing, Transfection, Staining, Flow Cytometry, Cytometry

13) Product Images from "Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells *"

Article Title: Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.246504

Effect of Bak and/or Bax knockdown on Cholix-induced apoptotic signals. A , cells (1 × 10 5 cells/well) in a 12-well dish were grown for 24 h, and silencing of bak alone, bax alone, or bak / bax gene was performed with non-targeting control ( NC ), Bak, or Bax siRNA as described under “Experimental Procedures.” 48 h after transfection, cells were incubated with wild-type ( WT ) or mutant ( MT ) Cholix (10 μg/ml) for 18 h. Reduction of Bak or Bax protein level in total cell lysate ( TCL ) was confirmed by Western blotting with anti-Bak or anti-Bax antibody. Cytochrome c release was determined as described under “Experimental Procedures.” Cholix-induced caspases cleavage was determined by Western blotting with anti-cleaved caspase-3, -7, or -9 antibodies. Mean values were calculated on relative band intensity in three separate experiments. B , the cells transfected with the siRNA overnight (1 × 10 4 cells/well) were plated in a 96-well dish, grown overnight, and then treated with Cholix (10 μg/ml) for 24 h. Cell viability was determined by using the Cell Counting kit as described under “Experimental Procedures.” Data are the means ± S.D. value from three separate experiments. The Student's t test was used for comparisons with the non-targeting control siRNA-transfected samples incubated with Cholix.
Figure Legend Snippet: Effect of Bak and/or Bax knockdown on Cholix-induced apoptotic signals. A , cells (1 × 10 5 cells/well) in a 12-well dish were grown for 24 h, and silencing of bak alone, bax alone, or bak / bax gene was performed with non-targeting control ( NC ), Bak, or Bax siRNA as described under “Experimental Procedures.” 48 h after transfection, cells were incubated with wild-type ( WT ) or mutant ( MT ) Cholix (10 μg/ml) for 18 h. Reduction of Bak or Bax protein level in total cell lysate ( TCL ) was confirmed by Western blotting with anti-Bak or anti-Bax antibody. Cytochrome c release was determined as described under “Experimental Procedures.” Cholix-induced caspases cleavage was determined by Western blotting with anti-cleaved caspase-3, -7, or -9 antibodies. Mean values were calculated on relative band intensity in three separate experiments. B , the cells transfected with the siRNA overnight (1 × 10 4 cells/well) were plated in a 96-well dish, grown overnight, and then treated with Cholix (10 μg/ml) for 24 h. Cell viability was determined by using the Cell Counting kit as described under “Experimental Procedures.” Data are the means ± S.D. value from three separate experiments. The Student's t test was used for comparisons with the non-targeting control siRNA-transfected samples incubated with Cholix.

Techniques Used: Transfection, Incubation, Mutagenesis, Western Blot, Cell Counting

14) Product Images from "Genetic Control of Translesion Synthesis on Leading and Lagging DNA Strands in Plasmids Derived from Epstein-Barr Virus in Human Cells"

Article Title: Genetic Control of Translesion Synthesis on Leading and Lagging DNA Strands in Plasmids Derived from Epstein-Barr Virus in Human Cells

Journal: mBio

doi: 10.1128/mBio.00271-12

Assay for determining the genetic control of TLS on the leading and lagging strands of an EBV origin-based plasmid. (A) The target 16-mer sequence containing a cis- syn TT dimer (T^T) is shown at the top. The sequence of the N-terminal part of the lacZ ′ gene in the pBSA vector (leading strand), including the TT dimer, is shown. (B) Strategy for TLS. In the duplex plasmid, the DNA strand containing the TT dimer carries the wild-type kanamycin resistance gene ( kan + ) so that TLS opposite the UV lesion will result in a blue colony on LB/Kan plates containing IPTG and X-Gal. (C) Assay for TLS and for determining replication efficiency of damage-containing plasmids in siRNA-treated human cells. The purified DNA lesion-containing plasmid, undamaged pCDNA3.1-Zeocin plasmid, and siRNA are cotransfected into human cells that have been pretreated with siRNA for 48 h. After 30 h incubation, the rescued plasmid DNA is treated with DpnI to remove any unreplicated plasmid, and then transformed into XL-1 Blue E. coli cells. TLS frequency is determined from the frequency of blue colonies among kan + colonies. The replication efficiency of undamaged EBV plasmid relative to that of the zeocin resistance plasmid was determined by the number of colonies that grew on LB/Amp plates, indicative of the EBV plasmid, and the number of colonies that grew on LB/Zeo plates, indicative of the zeocin plasmid.
Figure Legend Snippet: Assay for determining the genetic control of TLS on the leading and lagging strands of an EBV origin-based plasmid. (A) The target 16-mer sequence containing a cis- syn TT dimer (T^T) is shown at the top. The sequence of the N-terminal part of the lacZ ′ gene in the pBSA vector (leading strand), including the TT dimer, is shown. (B) Strategy for TLS. In the duplex plasmid, the DNA strand containing the TT dimer carries the wild-type kanamycin resistance gene ( kan + ) so that TLS opposite the UV lesion will result in a blue colony on LB/Kan plates containing IPTG and X-Gal. (C) Assay for TLS and for determining replication efficiency of damage-containing plasmids in siRNA-treated human cells. The purified DNA lesion-containing plasmid, undamaged pCDNA3.1-Zeocin plasmid, and siRNA are cotransfected into human cells that have been pretreated with siRNA for 48 h. After 30 h incubation, the rescued plasmid DNA is treated with DpnI to remove any unreplicated plasmid, and then transformed into XL-1 Blue E. coli cells. TLS frequency is determined from the frequency of blue colonies among kan + colonies. The replication efficiency of undamaged EBV plasmid relative to that of the zeocin resistance plasmid was determined by the number of colonies that grew on LB/Amp plates, indicative of the EBV plasmid, and the number of colonies that grew on LB/Zeo plates, indicative of the zeocin plasmid.

Techniques Used: Plasmid Preparation, Sequencing, Purification, Incubation, Transformation Assay

15) Product Images from "Relaxin Protects Rat Lungs from Ischemia-Reperfusion Injury via Inducible NO Synthase: Role of ERK-1/2, PI3K, and Forkhead Transcription Factor FKHRL1"

Article Title: Relaxin Protects Rat Lungs from Ischemia-Reperfusion Injury via Inducible NO Synthase: Role of ERK-1/2, PI3K, and Forkhead Transcription Factor FKHRL1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0075592

FKHRL1 is essential for relaxin signaling towards iNOS. Primary rat pulmonary artery endothelial cells (RPAEC, left side) and rat pulmonary artery smooth muscle cells (RPASMC, right side) were processed at baseline, after 90 min hypoxia (5% oxygen), or after another 90 min of normoxia (n = 5 each) in order to determine iNOS activity. Experiments were carried out in the presence of vehicle (control), 5 nM of relaxin, the ERK-1/2 inhibitor PD-98059 (PD) (50 µmol/l), the PI3K inhibitor wortmannin (WM) (100 nM), and combinations thereof. Prior to experiments, cells had been transfected with scrambled siRNA (control), FOXB2 siRNA, or FKHRL1 siRNA. While both transfection with scrambled siRNA and knock-down of non-related forkhead factor, FOXB2, had no influence knock-down of FKHRL-1 abolished the susceptibility of relaxin’s effect towards PI3K inhibition in ischemia and equalized the extent of iNOS induction in reperfusion. P
Figure Legend Snippet: FKHRL1 is essential for relaxin signaling towards iNOS. Primary rat pulmonary artery endothelial cells (RPAEC, left side) and rat pulmonary artery smooth muscle cells (RPASMC, right side) were processed at baseline, after 90 min hypoxia (5% oxygen), or after another 90 min of normoxia (n = 5 each) in order to determine iNOS activity. Experiments were carried out in the presence of vehicle (control), 5 nM of relaxin, the ERK-1/2 inhibitor PD-98059 (PD) (50 µmol/l), the PI3K inhibitor wortmannin (WM) (100 nM), and combinations thereof. Prior to experiments, cells had been transfected with scrambled siRNA (control), FOXB2 siRNA, or FKHRL1 siRNA. While both transfection with scrambled siRNA and knock-down of non-related forkhead factor, FOXB2, had no influence knock-down of FKHRL-1 abolished the susceptibility of relaxin’s effect towards PI3K inhibition in ischemia and equalized the extent of iNOS induction in reperfusion. P

Techniques Used: Activity Assay, Transfection, Inhibition

16) Product Images from "Quantitative proteomics identifies a Dab2/integrin module regulating cell migration"

Article Title: Quantitative proteomics identifies a Dab2/integrin module regulating cell migration

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200812160

Steady-state surface levels of integrins α1 and β1 but not αv increase in Dab2-deficient cells. (A) Fixed, nonpermeabilized control and Dab2-deficient HeLa cells were analyzed by FACS. (B) Mean and standard error of fluorescent intensity from four independent experiments are shown. (C) FACS analysis of fixed, permeabilized HeLa cells shows that total integrin β1 only increases slightly on Dab2 removal. RT-PCR for integrin β1 and actin mRNA shows equal mRNA levels in control and Dab2-deficient cells. Mean and standard error from five independent experiments are shown. (D) HFFs treated with control or Dab2 siRNA. Western blot showing decreased Dab2 protein and FACS results showing increased surface integrin β1 (mean and standard error of two independent experiments). Data for total integrin β1 level in Dab2-deficient HFFs are shown in Fig. S1 B . (B and D) Dashed lines indicate the control levels. #, P
Figure Legend Snippet: Steady-state surface levels of integrins α1 and β1 but not αv increase in Dab2-deficient cells. (A) Fixed, nonpermeabilized control and Dab2-deficient HeLa cells were analyzed by FACS. (B) Mean and standard error of fluorescent intensity from four independent experiments are shown. (C) FACS analysis of fixed, permeabilized HeLa cells shows that total integrin β1 only increases slightly on Dab2 removal. RT-PCR for integrin β1 and actin mRNA shows equal mRNA levels in control and Dab2-deficient cells. Mean and standard error from five independent experiments are shown. (D) HFFs treated with control or Dab2 siRNA. Western blot showing decreased Dab2 protein and FACS results showing increased surface integrin β1 (mean and standard error of two independent experiments). Data for total integrin β1 level in Dab2-deficient HFFs are shown in Fig. S1 B . (B and D) Dashed lines indicate the control levels. #, P

Techniques Used: FACS, Reverse Transcription Polymerase Chain Reaction, Western Blot

Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in Fig. 2 B . (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P
Figure Legend Snippet: Effect of ARH, AP2, clathrin, or Numb depletion on surface integrin levels. (A–C) HeLa cells were treated with various siRNAs and then fixed and analyzed by FACS with anti-integrin or anti-TfnR antibody. Mean values and standard errors from at least three independent experiments are shown. Dashed lines indicate the control levels. (A) ARH has no effect; Dab2 depletion increases surface α1 and β1; AP2 or clathrin depletion significantly increases surface α1, α5, and β1. (B) Numb depletion has a bigger effect on surface α5 than on α1 or β1. In contrast, Dab2 depletion has a bigger effect on surface α1 and β1 than on α5. The Dab2 data are the same as those shown in Fig. 2 B . (C) Effects of separate or combined removal of Dab2 and Numb. Combined removal of Dab2 and Numb caused little further increase in integrin β1. (A and C) Western blot analysis of HeLa total lysates demonstrates that target proteins ARH, Dab2, AP2, clathrin heavy chain (CHC), and Numb were greatly reduced by siRNA transfection. As a control, extracellular signal-regulated kinase (ERK) levels remained constant. (B and C) #, P

Techniques Used: FACS, Western Blot, Transfection

17) Product Images from "The Amelioration of N-Acetyl-p-Benzoquinone Imine Toxicity by Ginsenoside Rg3: The Role of Nrf2-Mediated Detoxification and Mrp1/Mrp3 Transports"

Article Title: The Amelioration of N-Acetyl-p-Benzoquinone Imine Toxicity by Ginsenoside Rg3: The Role of Nrf2-Mediated Detoxification and Mrp1/Mrp3 Transports

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2013/957947

The Nrf2-mediated GCL gene expression by Rg3. (a) H4IIE cells were transiently transfected with siRNA directed against Nrf2 or nontargeted siRNA and subsequently treated with Rg3 (3 μ g/mL) or vehicle for 12 h. The representative blots of mRNA after Rg3 treatment were assessed by RT-PCR (left). To confirm gene knockdown, the Nrf2 levels 48 h after siRNA transfection were determined in the cell lysates by western blotting. GCLC and GCLM protein expression in the total cell lysates were detected in cells stimulated with Rg3 (3 μ g/mL) for 24 h (right). β -actin was used as the loading control. (b) MEFs from wild-type (Nrf2+/+) or Nrf2-deficient mice (Nrf2−/−) were treated with Rg3 (3 μ g/mL) or vehicle for 12 h. The mRNA levels (left) and protein expression (right) for the genes were analyzed as described above (a). All experiments were performed using independent mRNA and lysate samples. Each value represents the average with at least three separate experiments.
Figure Legend Snippet: The Nrf2-mediated GCL gene expression by Rg3. (a) H4IIE cells were transiently transfected with siRNA directed against Nrf2 or nontargeted siRNA and subsequently treated with Rg3 (3 μ g/mL) or vehicle for 12 h. The representative blots of mRNA after Rg3 treatment were assessed by RT-PCR (left). To confirm gene knockdown, the Nrf2 levels 48 h after siRNA transfection were determined in the cell lysates by western blotting. GCLC and GCLM protein expression in the total cell lysates were detected in cells stimulated with Rg3 (3 μ g/mL) for 24 h (right). β -actin was used as the loading control. (b) MEFs from wild-type (Nrf2+/+) or Nrf2-deficient mice (Nrf2−/−) were treated with Rg3 (3 μ g/mL) or vehicle for 12 h. The mRNA levels (left) and protein expression (right) for the genes were analyzed as described above (a). All experiments were performed using independent mRNA and lysate samples. Each value represents the average with at least three separate experiments.

Techniques Used: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mouse Assay

The effect of Nrf2 loss on the Mrp family gene expressions by Rg3. (a) H4IIE cells were transiently transfected with Nrf2-specific siRNA or control siRNA and subsequently treated with Rg3 (3 μ g/mL) or vehicle for 12 h. The representative blots of mRNA after Rg3 treatment were assessed by RT-PCR. β -actin was used as the loading control. (b) MEFs from wild-type (Nrf2+/+) or Nrf2-deficient mice (Nrf2−/−) were treated with Rg3 (3 μ g/mL) or vehicle for 12 h. The mRNA levels for the genes were analyzed as described above (a). The multiple analyses of 3 independent experiments were assessed by scanning densitometry. Each value represents the mean ± S.E. (** P
Figure Legend Snippet: The effect of Nrf2 loss on the Mrp family gene expressions by Rg3. (a) H4IIE cells were transiently transfected with Nrf2-specific siRNA or control siRNA and subsequently treated with Rg3 (3 μ g/mL) or vehicle for 12 h. The representative blots of mRNA after Rg3 treatment were assessed by RT-PCR. β -actin was used as the loading control. (b) MEFs from wild-type (Nrf2+/+) or Nrf2-deficient mice (Nrf2−/−) were treated with Rg3 (3 μ g/mL) or vehicle for 12 h. The mRNA levels for the genes were analyzed as described above (a). The multiple analyses of 3 independent experiments were assessed by scanning densitometry. Each value represents the mean ± S.E. (** P

Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

The GSH levels after NAPQI treatment with Rg3 in Nrf2-deficient cells. H4IIE cells were transiently transfected with siRNA directed against Nrf2 or control siRNA and subsequently treated with Rg3 (3 μ g/mL) or vehicle for 24 h. Then, cells were incubated with NAPQI (200 μ M) for 2 h. The GSH level was determined in the cell homogenates. All experiments were performed in quadruplicate. Each value represents the mean ± S.E. (* P
Figure Legend Snippet: The GSH levels after NAPQI treatment with Rg3 in Nrf2-deficient cells. H4IIE cells were transiently transfected with siRNA directed against Nrf2 or control siRNA and subsequently treated with Rg3 (3 μ g/mL) or vehicle for 24 h. Then, cells were incubated with NAPQI (200 μ M) for 2 h. The GSH level was determined in the cell homogenates. All experiments were performed in quadruplicate. Each value represents the mean ± S.E. (* P

Techniques Used: Transfection, Incubation

18) Product Images from "Lysophosphatidic Acid Acyltransferase Beta Regulates mTOR Signaling"

Article Title: Lysophosphatidic Acid Acyltransferase Beta Regulates mTOR Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0078632

Knockdown of LPAAT-β protein expression results in inhibition of anchorage-dependent proliferation of pancreatic cancer cells. Inhibition of proliferation in (A) AsPC-1 cells transfected with 10 nM siRNA to LPAAT-β (LP-1) and non-targeting (nt) control; (B) AsPC-1 cells transfected for 72 hr with 25 nM non-targeting siRNA, LPAAT-β siRNA (LP-1, LP-2, LP-4), or pooled KRas siRNA; (C) Panc-1 cells transfected for 72 hr with 10 nM and 25 nM non-targeting or LPAAT-β siRNA; (D) MiaPaCa2 cells transfected for 72 hr with 25 nM non-targeting, LPAAT-β, or pooled KRas siRNA. Experiments are representative of at least three replicates. Significance was determined using Student’s t-test, * p
Figure Legend Snippet: Knockdown of LPAAT-β protein expression results in inhibition of anchorage-dependent proliferation of pancreatic cancer cells. Inhibition of proliferation in (A) AsPC-1 cells transfected with 10 nM siRNA to LPAAT-β (LP-1) and non-targeting (nt) control; (B) AsPC-1 cells transfected for 72 hr with 25 nM non-targeting siRNA, LPAAT-β siRNA (LP-1, LP-2, LP-4), or pooled KRas siRNA; (C) Panc-1 cells transfected for 72 hr with 10 nM and 25 nM non-targeting or LPAAT-β siRNA; (D) MiaPaCa2 cells transfected for 72 hr with 25 nM non-targeting, LPAAT-β, or pooled KRas siRNA. Experiments are representative of at least three replicates. Significance was determined using Student’s t-test, * p

Techniques Used: Expressing, Inhibition, Transfection

19) Product Images from "Fibroblast Growth Factor Receptors as Novel Therapeutic Targets in SNF5-Deleted Malignant Rhabdoid Tumors"

Article Title: Fibroblast Growth Factor Receptors as Novel Therapeutic Targets in SNF5-Deleted Malignant Rhabdoid Tumors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077652

SNF5 loss of function induces FGFR2 expression in human fibroblasts. (A) Effect of siRNA-mediated knockdown of SNF5 on FGFR2 expression in BJ cells. SNF5 and FGFR2 expression levels were analyzed by qRT-PCR at 72 h post siRNA transfection. Expression is shown as relative levels to cells transfected with non-targeting control siRNA and is given as average with SEM (n≥3). E xpression values were normalized to GAPDH mRNA copies. (B) Immunoblot analysis of FGFR2 expression upon knockdown of SNF5 in BJ cells as described in (A). β-Tubulin expression was used to monitor equal loading. (C) Effect of siRNA-mediated knockdown of BRG1 on FGFR2 expression in BJ cells as described in (A).
Figure Legend Snippet: SNF5 loss of function induces FGFR2 expression in human fibroblasts. (A) Effect of siRNA-mediated knockdown of SNF5 on FGFR2 expression in BJ cells. SNF5 and FGFR2 expression levels were analyzed by qRT-PCR at 72 h post siRNA transfection. Expression is shown as relative levels to cells transfected with non-targeting control siRNA and is given as average with SEM (n≥3). E xpression values were normalized to GAPDH mRNA copies. (B) Immunoblot analysis of FGFR2 expression upon knockdown of SNF5 in BJ cells as described in (A). β-Tubulin expression was used to monitor equal loading. (C) Effect of siRNA-mediated knockdown of BRG1 on FGFR2 expression in BJ cells as described in (A).

Techniques Used: Expressing, Quantitative RT-PCR, Transfection

20) Product Images from "Downregulation of S100A4 expression by RNA interference suppresses cell growth and invasion in human colorectal cancer cells"

Article Title: Downregulation of S100A4 expression by RNA interference suppresses cell growth and invasion in human colorectal cancer cells

Journal: Oncology Reports

doi: 10.3892/or.2011.1598

S100A4 gene knockdown by siRNA transfection in SW620 cells. (A) By real-time PCR, S100A4 mRNA expression was significantly suppressed at 36 h post transfection of S100A4 siRNA into SW620 cells compared with mock and siControl of SW620 cells. Data represent the mean ± standard deviation (SD) of three independent experiments. * Specific comparison between the siS100A4 group and mock control, siControl group (P
Figure Legend Snippet: S100A4 gene knockdown by siRNA transfection in SW620 cells. (A) By real-time PCR, S100A4 mRNA expression was significantly suppressed at 36 h post transfection of S100A4 siRNA into SW620 cells compared with mock and siControl of SW620 cells. Data represent the mean ± standard deviation (SD) of three independent experiments. * Specific comparison between the siS100A4 group and mock control, siControl group (P

Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

S100A4 siRNA inhibited the proliferation of SW620 cells in vitro . Cell proliferation of SW620 cells transfected with mock control, siControl and siS100A4 was analyzed by the Cell Counting kit-8 assay. Cells were seeded in 96-well culture plates (1×10 5 cells/well). Cell proliferation was determined daily for 7 days in all. The absorbance was read at 490 nm by a spectrophotometer microplate reader. Data represent the mean ± standard deviation (SD) of six independent experiments. * Specific comparison between the siS100A4 group and the mock control, siControl group at Days 3, 4, 5, 6 and 7 of the time course (P
Figure Legend Snippet: S100A4 siRNA inhibited the proliferation of SW620 cells in vitro . Cell proliferation of SW620 cells transfected with mock control, siControl and siS100A4 was analyzed by the Cell Counting kit-8 assay. Cells were seeded in 96-well culture plates (1×10 5 cells/well). Cell proliferation was determined daily for 7 days in all. The absorbance was read at 490 nm by a spectrophotometer microplate reader. Data represent the mean ± standard deviation (SD) of six independent experiments. * Specific comparison between the siS100A4 group and the mock control, siControl group at Days 3, 4, 5, 6 and 7 of the time course (P

Techniques Used: In Vitro, Transfection, Cell Counting, Spectrophotometry, Standard Deviation

21) Product Images from "Vitamin E ?-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism"

Article Title: Vitamin E ?-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052526

p27 Kip1 is required for δ-tocotrienol-induced G 1 arrest. A, p27 Kip1 siRNA attenuates δ-tocotrienol-mediated growth suppression in human MIAPaCa-2 pancreatic cancer cells. After transfection with p27 Kip1 siRNA or with noncoding siRNA for 24 hours, MIAPaCa-2 cells were incubated with fresh medium containing either δ-tocotrienol (IC 50 ) or vehicle for an additional 24 hours. Cells were taken from culture and divided into 2 aliquots. Immunoblots demonstrate inhibited p27 Kip1 expression with siRNA p27 Kip1 and rescue from inhibition of proliferation in siRNA p27 Kip1 -pretreated cells. B, MIAPaCa-2 cells expressing stable shRNA p27 Kip1 are protected from the growth inhibitory effects of δ-tocotrienol. Stable MIAPaCa-2 cells expressing shRNAp27 Kip1 or empty vector were treated with increasing concentrations of δ-tocotrienol or vehicle for 24 hours, with proliferation determined by MTT assay. MIAPaCa-2 cells expressing shRNAp27 Kip1 demonstrate resistance to growth inhibitory effects of δ-tocotrienol. C, p27 Kip1 knockout cells attenuate δ-tocotrienol-mediated growth inhibitory effects. Stable MEF p27 Kip1 (−/−) and MEF p27 Kip1 (+/+) were plated and incubated with either δ-tocotrienol at the indicated doses or with vehicle for 48 hours. Cell cultures were then collected in 2 aliquots and analyzed as reported previously for siRNA p27 Kip1 -treated cells. In the absence of p27 Kip1 expression, δ-tocotrienol exerts minimal growth inhibitory effects in mouse epithelial cells.
Figure Legend Snippet: p27 Kip1 is required for δ-tocotrienol-induced G 1 arrest. A, p27 Kip1 siRNA attenuates δ-tocotrienol-mediated growth suppression in human MIAPaCa-2 pancreatic cancer cells. After transfection with p27 Kip1 siRNA or with noncoding siRNA for 24 hours, MIAPaCa-2 cells were incubated with fresh medium containing either δ-tocotrienol (IC 50 ) or vehicle for an additional 24 hours. Cells were taken from culture and divided into 2 aliquots. Immunoblots demonstrate inhibited p27 Kip1 expression with siRNA p27 Kip1 and rescue from inhibition of proliferation in siRNA p27 Kip1 -pretreated cells. B, MIAPaCa-2 cells expressing stable shRNA p27 Kip1 are protected from the growth inhibitory effects of δ-tocotrienol. Stable MIAPaCa-2 cells expressing shRNAp27 Kip1 or empty vector were treated with increasing concentrations of δ-tocotrienol or vehicle for 24 hours, with proliferation determined by MTT assay. MIAPaCa-2 cells expressing shRNAp27 Kip1 demonstrate resistance to growth inhibitory effects of δ-tocotrienol. C, p27 Kip1 knockout cells attenuate δ-tocotrienol-mediated growth inhibitory effects. Stable MEF p27 Kip1 (−/−) and MEF p27 Kip1 (+/+) were plated and incubated with either δ-tocotrienol at the indicated doses or with vehicle for 48 hours. Cell cultures were then collected in 2 aliquots and analyzed as reported previously for siRNA p27 Kip1 -treated cells. In the absence of p27 Kip1 expression, δ-tocotrienol exerts minimal growth inhibitory effects in mouse epithelial cells.

Techniques Used: Transfection, Incubation, Western Blot, Expressing, Inhibition, shRNA, Plasmid Preparation, MTT Assay, Knock-Out

δ-Tocotrienol (DT3) regulation of p27 Kip1 protein expression at the transcription level. A, MIAPaca-2 cells were treated with δ-tocotrienol at a predetermined IC 50 and then with cyclohexamide to block protein synthesis. p27 Kip1 turnover rate was examined by Western blot. B, densitometry plot for panel A using β-actin for density control, showing similar rates of degradation by δ-tocotrienol. C, RT-PCR confirms upregulation of p27 Kip1 at the mRNA level. MIAPaCa-2 cells were treated with δ-tocotrienol for 24 hours, and p27 Kip1 mRNA expression level was determined by RT-PCR at different amplification cycles. Representative result at cycle 40 is shown. D, activation of p27 Kip1 promoter by δ-tocotrienol. MIAPaCa-2 cells were transfected with 5′-deletion mutants of the mouse p27 Kip1 promoter luciferase reporter. After 24-hour transfection, cells were treated with δ-tocotrienol (IC 50 ) or vehicle for an additional 24 hours, and luciferase activity was determined. Deletion analysis of the mouse p27 Kip1 promoter suggests that the region between 326 and 615 contains sequences necessary for significant response to δ-tocotrienol in p27 Kip1 reporter assays. E, illustration of 5′ deletion mutants of the mouse p27 Kip1 promoter luciferase reporter. Sequence search of this region revealed several putative E2F-1 binding sites (TTTGGCTA, GCGCGGAG, GCGCCGAG) as demonstrated in the shaded area of the deletion mutant constructs. F , immunoblot showing suppressed E2F-1 expression using siRNA E2F-1, G , attenuated δ-tocotrienol mediated effects on the transfected full-length p27 Kip1 promoter activity using a luciferase reporter assay.
Figure Legend Snippet: δ-Tocotrienol (DT3) regulation of p27 Kip1 protein expression at the transcription level. A, MIAPaca-2 cells were treated with δ-tocotrienol at a predetermined IC 50 and then with cyclohexamide to block protein synthesis. p27 Kip1 turnover rate was examined by Western blot. B, densitometry plot for panel A using β-actin for density control, showing similar rates of degradation by δ-tocotrienol. C, RT-PCR confirms upregulation of p27 Kip1 at the mRNA level. MIAPaCa-2 cells were treated with δ-tocotrienol for 24 hours, and p27 Kip1 mRNA expression level was determined by RT-PCR at different amplification cycles. Representative result at cycle 40 is shown. D, activation of p27 Kip1 promoter by δ-tocotrienol. MIAPaCa-2 cells were transfected with 5′-deletion mutants of the mouse p27 Kip1 promoter luciferase reporter. After 24-hour transfection, cells were treated with δ-tocotrienol (IC 50 ) or vehicle for an additional 24 hours, and luciferase activity was determined. Deletion analysis of the mouse p27 Kip1 promoter suggests that the region between 326 and 615 contains sequences necessary for significant response to δ-tocotrienol in p27 Kip1 reporter assays. E, illustration of 5′ deletion mutants of the mouse p27 Kip1 promoter luciferase reporter. Sequence search of this region revealed several putative E2F-1 binding sites (TTTGGCTA, GCGCGGAG, GCGCCGAG) as demonstrated in the shaded area of the deletion mutant constructs. F , immunoblot showing suppressed E2F-1 expression using siRNA E2F-1, G , attenuated δ-tocotrienol mediated effects on the transfected full-length p27 Kip1 promoter activity using a luciferase reporter assay.

Techniques Used: Expressing, Blocking Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Activation Assay, Transfection, Luciferase, Activity Assay, Sequencing, Binding Assay, Mutagenesis, Construct, Reporter Assay

22) Product Images from "Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy"

Article Title: Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0079795

Presence of both Akt and Phafin2 are required for induction of autophagy. A–D . Phafin2-siRNA transfected macrophages showed no inhibition on initial uptake of fluorescent bacteria ( A–B , top panels). However, after HBSS treatment to induce autophagy, Phafin2-siRNAs transfected cells inhibited not only elimination of fluorescent bacteria ( A–B , middle panels), but also induction of autophagy ( A–B , bottom panels), which is reversible by re-introduction of human Phafin2 ( C–D , bottom panels). Note that human Phafin2 is resistant for mouse Phafin2-siRNA. Quantification of LC3 puncta per cell with statistical analysis by Student's t -test is shown on the right side. White scale bar represents 10 µm. E–H . Using J774.1 murine macrophages, LPS-induced lysosomal accumulation of Akt was eliminated by Phafin2-siRNAs ( E–F ), which is associated with inhibition of autophagy ( I–J , lower panels). The observations are reversible by re-introduction of human Phafin2 ( G–H and K–L , lower panels). Quantification of the percent of colocalization area of Akt with LAMP2 with statistical analysis by Student's t -test is shown on the right side. I–L . Phafin2-siRNA transfected macrophages inhibited LPS-induced autophagy determined by LC3 pancta with Phafin2 expression shown in inset (bottom panels, compare I and J ). Inhibition of autophagy by Phafin2-siRNAs can be reverted by re-introduction of human Phafin2, which is resistant for mouse Phafin2-siRNA (bottom panels, compare K and L ). Quantification of LC3 puncta per cell with statistical analysis by Student's t -test is shown on the right side. M . LC3 immunoblot by the introduction of Phafin2-siRNA were shown. The percentage of autophagy inhibition out of three independent experiments was 53.3±21.4%. N–Q . Akt-siRNA ( fig. S5 ) transfected macrophages retained LPS-induced lysosomal translocation/accumulation of Phafin2 (upper panels). Akt-siRNA, however, inhibited LPS-induced autophagy determined by LC3 pancta with Akt expression shown in inset (bottom panels, compare O and Q ). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t -test were shown as a bar graph. R–U . Akt-siRNA ( fig. S5 ) transfected HeLa cells failed to induce autophagy determined by LC3 puncta on Ds-Red positive cells (inset). Further, inhibition of autophagy by Akt-siRNAs can be reversed by re-introduction of siRNA-resistant human Akt2 in HeLa cells ( U ). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t -test were shown as a bar graph. V . LC3 immunoblot by the introduction of Akt siRNA were shown. The percentage of autophagy inhibition (shown in the bar graph) was 45.4±11.2% out of three independent experiments.
Figure Legend Snippet: Presence of both Akt and Phafin2 are required for induction of autophagy. A–D . Phafin2-siRNA transfected macrophages showed no inhibition on initial uptake of fluorescent bacteria ( A–B , top panels). However, after HBSS treatment to induce autophagy, Phafin2-siRNAs transfected cells inhibited not only elimination of fluorescent bacteria ( A–B , middle panels), but also induction of autophagy ( A–B , bottom panels), which is reversible by re-introduction of human Phafin2 ( C–D , bottom panels). Note that human Phafin2 is resistant for mouse Phafin2-siRNA. Quantification of LC3 puncta per cell with statistical analysis by Student's t -test is shown on the right side. White scale bar represents 10 µm. E–H . Using J774.1 murine macrophages, LPS-induced lysosomal accumulation of Akt was eliminated by Phafin2-siRNAs ( E–F ), which is associated with inhibition of autophagy ( I–J , lower panels). The observations are reversible by re-introduction of human Phafin2 ( G–H and K–L , lower panels). Quantification of the percent of colocalization area of Akt with LAMP2 with statistical analysis by Student's t -test is shown on the right side. I–L . Phafin2-siRNA transfected macrophages inhibited LPS-induced autophagy determined by LC3 pancta with Phafin2 expression shown in inset (bottom panels, compare I and J ). Inhibition of autophagy by Phafin2-siRNAs can be reverted by re-introduction of human Phafin2, which is resistant for mouse Phafin2-siRNA (bottom panels, compare K and L ). Quantification of LC3 puncta per cell with statistical analysis by Student's t -test is shown on the right side. M . LC3 immunoblot by the introduction of Phafin2-siRNA were shown. The percentage of autophagy inhibition out of three independent experiments was 53.3±21.4%. N–Q . Akt-siRNA ( fig. S5 ) transfected macrophages retained LPS-induced lysosomal translocation/accumulation of Phafin2 (upper panels). Akt-siRNA, however, inhibited LPS-induced autophagy determined by LC3 pancta with Akt expression shown in inset (bottom panels, compare O and Q ). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t -test were shown as a bar graph. R–U . Akt-siRNA ( fig. S5 ) transfected HeLa cells failed to induce autophagy determined by LC3 puncta on Ds-Red positive cells (inset). Further, inhibition of autophagy by Akt-siRNAs can be reversed by re-introduction of siRNA-resistant human Akt2 in HeLa cells ( U ). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t -test were shown as a bar graph. V . LC3 immunoblot by the introduction of Akt siRNA were shown. The percentage of autophagy inhibition (shown in the bar graph) was 45.4±11.2% out of three independent experiments.

Techniques Used: Transfection, Inhibition, Expressing, Translocation Assay

23) Product Images from "Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy"

Article Title: Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0079795

Presence of both Akt and Phafin2 are required for induction of autophagy. A–D . Phafin2-siRNA transfected macrophages showed no inhibition on initial uptake of fluorescent bacteria ( A–B , top panels). However, after HBSS treatment to induce autophagy, Phafin2-siRNAs transfected cells inhibited not only elimination of fluorescent bacteria ( A–B , middle panels), but also induction of autophagy ( A–B , bottom panels), which is reversible by re-introduction of human Phafin2 ( C–D , bottom panels). Note that human Phafin2 is resistant for mouse Phafin2-siRNA. Quantification of LC3 puncta per cell with statistical analysis by Student's t -test is shown on the right side. White scale bar represents 10 µm. E–H . Using J774.1 murine macrophages, LPS-induced lysosomal accumulation of Akt was eliminated by Phafin2-siRNAs ( E–F ), which is associated with inhibition of autophagy ( I–J , lower panels). The observations are reversible by re-introduction of human Phafin2 ( G–H and K–L , lower panels). Quantification of the percent of colocalization area of Akt with LAMP2 with statistical analysis by Student's t -test is shown on the right side. I–L . Phafin2-siRNA transfected macrophages inhibited LPS-induced autophagy determined by LC3 pancta with Phafin2 expression shown in inset (bottom panels, compare I and J ). Inhibition of autophagy by Phafin2-siRNAs can be reverted by re-introduction of human Phafin2, which is resistant for mouse Phafin2-siRNA (bottom panels, compare K and L ). Quantification of LC3 puncta per cell with statistical analysis by Student's t -test is shown on the right side. M . LC3 immunoblot by the introduction of Phafin2-siRNA were shown. The percentage of autophagy inhibition out of three independent experiments was 53.3±21.4%. N–Q . Akt-siRNA ( fig. S5 ) transfected macrophages retained LPS-induced lysosomal translocation/accumulation of Phafin2 (upper panels). Akt-siRNA, however, inhibited LPS-induced autophagy determined by LC3 pancta with Akt expression shown in inset (bottom panels, compare O and Q ). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t -test were shown as a bar graph. R–U . Akt-siRNA ( fig. S5 ) transfected HeLa cells failed to induce autophagy determined by LC3 puncta on Ds-Red positive cells (inset). Further, inhibition of autophagy by Akt-siRNAs can be reversed by re-introduction of siRNA-resistant human Akt2 in HeLa cells ( U ). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t -test were shown as a bar graph. V . LC3 immunoblot by the introduction of Akt siRNA were shown. The percentage of autophagy inhibition (shown in the bar graph) was 45.4±11.2% out of three independent experiments.
Figure Legend Snippet: Presence of both Akt and Phafin2 are required for induction of autophagy. A–D . Phafin2-siRNA transfected macrophages showed no inhibition on initial uptake of fluorescent bacteria ( A–B , top panels). However, after HBSS treatment to induce autophagy, Phafin2-siRNAs transfected cells inhibited not only elimination of fluorescent bacteria ( A–B , middle panels), but also induction of autophagy ( A–B , bottom panels), which is reversible by re-introduction of human Phafin2 ( C–D , bottom panels). Note that human Phafin2 is resistant for mouse Phafin2-siRNA. Quantification of LC3 puncta per cell with statistical analysis by Student's t -test is shown on the right side. White scale bar represents 10 µm. E–H . Using J774.1 murine macrophages, LPS-induced lysosomal accumulation of Akt was eliminated by Phafin2-siRNAs ( E–F ), which is associated with inhibition of autophagy ( I–J , lower panels). The observations are reversible by re-introduction of human Phafin2 ( G–H and K–L , lower panels). Quantification of the percent of colocalization area of Akt with LAMP2 with statistical analysis by Student's t -test is shown on the right side. I–L . Phafin2-siRNA transfected macrophages inhibited LPS-induced autophagy determined by LC3 pancta with Phafin2 expression shown in inset (bottom panels, compare I and J ). Inhibition of autophagy by Phafin2-siRNAs can be reverted by re-introduction of human Phafin2, which is resistant for mouse Phafin2-siRNA (bottom panels, compare K and L ). Quantification of LC3 puncta per cell with statistical analysis by Student's t -test is shown on the right side. M . LC3 immunoblot by the introduction of Phafin2-siRNA were shown. The percentage of autophagy inhibition out of three independent experiments was 53.3±21.4%. N–Q . Akt-siRNA ( fig. S5 ) transfected macrophages retained LPS-induced lysosomal translocation/accumulation of Phafin2 (upper panels). Akt-siRNA, however, inhibited LPS-induced autophagy determined by LC3 pancta with Akt expression shown in inset (bottom panels, compare O and Q ). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t -test were shown as a bar graph. R–U . Akt-siRNA ( fig. S5 ) transfected HeLa cells failed to induce autophagy determined by LC3 puncta on Ds-Red positive cells (inset). Further, inhibition of autophagy by Akt-siRNAs can be reversed by re-introduction of siRNA-resistant human Akt2 in HeLa cells ( U ). Number of GFP-LC3 puncta per cells with statistical analysis by Student's t -test were shown as a bar graph. V . LC3 immunoblot by the introduction of Akt siRNA were shown. The percentage of autophagy inhibition (shown in the bar graph) was 45.4±11.2% out of three independent experiments.

Techniques Used: Transfection, Inhibition, Expressing, Translocation Assay

24) Product Images from "IFN-?-driven CCL2 Production Recruits Inflammatory Monocytes to Infection Site in Mice"

Article Title: IFN-?-driven CCL2 Production Recruits Inflammatory Monocytes to Infection Site in Mice

Journal: Mucosal immunology

doi: 10.1038/mi.2012.46

IFI-16 drives CCL2 production by way of type I IFN ( A ) THCE cells were transfected with control or siRNA specific to IFI-16 and infected with HSV-1. Twenty-four hours pi, mRNA transcript expression was evaluated for CCL2 and normalized to uninfected controls and the housekeeping gene β-actin. Results are expressed as the mean ± SEM. ( B ) STING −/− , CD118 −/− , and WT mice (n = 4–8) were infected with 1,000 PFU / eye. Forty-eight hours pi chemokine content was evaluated by suspension array and is presented as the mean pg / mg ± SEM. ( C ) Evaluation of the upstream promoter of CCL2 identified IFN (red) and NF-κB (green) responsive elements. ( D ) WT, MyD88 −/− , Trif −/− , and CD118 −/− mice were infected and NF-κB nuclear translocation was assessed 44 hours pi by Western blot and compared to uninfected (UI) controls. Image is representative of 2 independent experiments. ( E ) THCE cells (n = 9) were treated with rIFN-α and CCL2 mRNA transcript assessed at 6 or 12 hours post-treatment. Results were normalized to ddH 2 0 controls and the housekeeping gene β-actin. Tx, treatment; IU, units; Values are expressed as the mean ± SEM of 2–3 independent experiments. ( F ) To confirm IFN was responsible for driving CCL2, WT and CD118−/− mice were treated with PBS or 30,0000 U rIFN-α. Twenty-four hrs later, chemokine levels were assessed and are presented as the mean ± SEM of 3 independent experiments of 2–3 corneas / group. **, p
Figure Legend Snippet: IFI-16 drives CCL2 production by way of type I IFN ( A ) THCE cells were transfected with control or siRNA specific to IFI-16 and infected with HSV-1. Twenty-four hours pi, mRNA transcript expression was evaluated for CCL2 and normalized to uninfected controls and the housekeeping gene β-actin. Results are expressed as the mean ± SEM. ( B ) STING −/− , CD118 −/− , and WT mice (n = 4–8) were infected with 1,000 PFU / eye. Forty-eight hours pi chemokine content was evaluated by suspension array and is presented as the mean pg / mg ± SEM. ( C ) Evaluation of the upstream promoter of CCL2 identified IFN (red) and NF-κB (green) responsive elements. ( D ) WT, MyD88 −/− , Trif −/− , and CD118 −/− mice were infected and NF-κB nuclear translocation was assessed 44 hours pi by Western blot and compared to uninfected (UI) controls. Image is representative of 2 independent experiments. ( E ) THCE cells (n = 9) were treated with rIFN-α and CCL2 mRNA transcript assessed at 6 or 12 hours post-treatment. Results were normalized to ddH 2 0 controls and the housekeeping gene β-actin. Tx, treatment; IU, units; Values are expressed as the mean ± SEM of 2–3 independent experiments. ( F ) To confirm IFN was responsible for driving CCL2, WT and CD118−/− mice were treated with PBS or 30,0000 U rIFN-α. Twenty-four hrs later, chemokine levels were assessed and are presented as the mean ± SEM of 3 independent experiments of 2–3 corneas / group. **, p

Techniques Used: Transfection, Infection, Expressing, Mouse Assay, Translocation Assay, Western Blot

25) Product Images from "Protein Kinase C Epsilon and Genetic Networks in Osteosarcoma Metastasis"

Article Title: Protein Kinase C Epsilon and Genetic Networks in Osteosarcoma Metastasis

Journal: Cancers

doi: 10.3390/cancers5020372

( a ) PRKCε is not required for in vitro migration of highly metastatic M132 osteosarcoma cells. ( b ) Confirmation of PRKCε knockdown. ( c ) Quantification of migration by M132 cells. ( a ) Upper panel: photographs of M132 cells immediately after scratching, two days following transfection with PRKCε siRNA. Lower panel: matched photographs of M132 cells two days following scratching and four days following transfection with PRKCε siRNA. ( b ) Western blot verification of PRKCε knock-down two, three and four days following transfection with siRNA. ( c ) Quantification of migrated distance by M132 cells transfected with PRKCε siRNA and appropriate controls. Values are average ± standard error of three independent experiments.
Figure Legend Snippet: ( a ) PRKCε is not required for in vitro migration of highly metastatic M132 osteosarcoma cells. ( b ) Confirmation of PRKCε knockdown. ( c ) Quantification of migration by M132 cells. ( a ) Upper panel: photographs of M132 cells immediately after scratching, two days following transfection with PRKCε siRNA. Lower panel: matched photographs of M132 cells two days following scratching and four days following transfection with PRKCε siRNA. ( b ) Western blot verification of PRKCε knock-down two, three and four days following transfection with siRNA. ( c ) Quantification of migrated distance by M132 cells transfected with PRKCε siRNA and appropriate controls. Values are average ± standard error of three independent experiments.

Techniques Used: In Vitro, Migration, Transfection, Western Blot

26) Product Images from "Targeted Silencing of MART-1 Gene Expression by RNA Interference Enhances the Migration Ability of Uveal Melanoma Cells"

Article Title: Targeted Silencing of MART-1 Gene Expression by RNA Interference Enhances the Migration Ability of Uveal Melanoma Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms140715092

Targeted silencing of MART-1 gene expression by small interfering RNA. ( A ) Results of RT-PCR detection of MART-1 expression in SP6.5 cells transfected with BLANK, siNC, or siMart-1 for 24 h; ( B ) Quantitative real-time PCR analysis of MART-1 gene transcripts in SP6.5 cells. The experiment was performed after 24 h of siMart-1 (100 nmol/L) transfection. GAPDH was used as the internal control. ★★ p
Figure Legend Snippet: Targeted silencing of MART-1 gene expression by small interfering RNA. ( A ) Results of RT-PCR detection of MART-1 expression in SP6.5 cells transfected with BLANK, siNC, or siMart-1 for 24 h; ( B ) Quantitative real-time PCR analysis of MART-1 gene transcripts in SP6.5 cells. The experiment was performed after 24 h of siMart-1 (100 nmol/L) transfection. GAPDH was used as the internal control. ★★ p

Techniques Used: Expressing, Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Transfection, Real-time Polymerase Chain Reaction

27) Product Images from "Human Apolipoprotein A-I Is Associated with Dengue Virus and Enhances Virus Infection through SR-BI"

Article Title: Human Apolipoprotein A-I Is Associated with Dengue Virus and Enhances Virus Infection through SR-BI

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070390

Down-regulation of SR-BI reduces DV infection in Huh-7 cells. (A) U937 cells and (B) Huh-7 cells were transfected with siRNA CTL or siRNA SR-BI. At 2 dpt, U937 cells and Huh-7 cells were infected with DV/SFM or DV/HSM at an MOI of 0.1. Total RNA was extracted at 1 dpi. Viral replication was measured by real-time PCR. The results represent the average standard deviation of three independent experiments. NS, no significance; **p
Figure Legend Snippet: Down-regulation of SR-BI reduces DV infection in Huh-7 cells. (A) U937 cells and (B) Huh-7 cells were transfected with siRNA CTL or siRNA SR-BI. At 2 dpt, U937 cells and Huh-7 cells were infected with DV/SFM or DV/HSM at an MOI of 0.1. Total RNA was extracted at 1 dpi. Viral replication was measured by real-time PCR. The results represent the average standard deviation of three independent experiments. NS, no significance; **p

Techniques Used: Infection, Transfection, CTL Assay, Real-time Polymerase Chain Reaction, Standard Deviation

28) Product Images from "Ribonucleotides Misincorporated into DNA Act as Strand-Discrimination Signals in Eukaryotic Mismatch Repair"

Article Title: Ribonucleotides Misincorporated into DNA Act as Strand-Discrimination Signals in Eukaryotic Mismatch Repair

Journal: Molecular Cell

doi: 10.1016/j.molcel.2013.03.019

RNase H2-Activated MMR in Human and Mouse Nuclear Extracts (A) RNASEH2A knockdown decreases MMR activity on the rC-T/G heteroduplex. (Left panels) Efficiency of the mismatch repair reaction at 5 and 15 min time points in extracts of 293 cells transiently transfected with the indicated siRNAs. The figure shows an agarose gel image (GelRed) and autoradiograph ( 32 P) of a representative experiment. (Top right) Plot of data from the 15 min time points of three independent experiments, with error bars representing standard deviation from the mean. (Bottom right) Western blot showing siRNA-mediated knockdown efficiency of RNASEH2A in 293 cells. Luc, siRNA against luciferase control; H1, H2a, H1+H2a, siRNAs against RNase H1, H2a, or both. (B) Immunodepletion of RNase H2 decreases MMR activity on the T/G-rC heteroduplex. (Left panels) MMR assay carried out in extracts of 293T cells immunodepleted of RNase H2. The figure shows an agarose gel image (GelRed) and autoradiograph ( 32 P) of 5 and 15 min time points of a representative experiment. (Top right) Plot of data from the 15 min time points of three independent experiments, with error bars representing standard deviation from the mean. (Bottom right) Immunodepletion of RNase H2 and supplementation of 293T extracts with recombinant MutLα. This western blot shows the efficiency of the immunodepletion procedure and the amounts of recombinant MLH1 relative to endogenous MSH2 levels. (C) Extracts of RNase H2 knockout mouse embryonal fibroblasts display decreased MMR activity on the T/G-rC heteroduplex at the 15 and 30 min time points. (Left panel) An agarose gel image (GelRed) and autoradiograph ( 32 P) of a representative experiment. (Top right) Plot of data from the 30 min time points of three independent experiments, with error bars representing standard deviation from the mean. (Bottom right) Western blot showing the amount of RNASEH2A in the MEFs as well as the amount of recombinant RNase H2 protein added to restore the MMR defect. MSH2 visualized with an anti-MSH2 antibody served as the loading control in the above experiments. M, size marker (1 kb ladder, New England BioLabs).
Figure Legend Snippet: RNase H2-Activated MMR in Human and Mouse Nuclear Extracts (A) RNASEH2A knockdown decreases MMR activity on the rC-T/G heteroduplex. (Left panels) Efficiency of the mismatch repair reaction at 5 and 15 min time points in extracts of 293 cells transiently transfected with the indicated siRNAs. The figure shows an agarose gel image (GelRed) and autoradiograph ( 32 P) of a representative experiment. (Top right) Plot of data from the 15 min time points of three independent experiments, with error bars representing standard deviation from the mean. (Bottom right) Western blot showing siRNA-mediated knockdown efficiency of RNASEH2A in 293 cells. Luc, siRNA against luciferase control; H1, H2a, H1+H2a, siRNAs against RNase H1, H2a, or both. (B) Immunodepletion of RNase H2 decreases MMR activity on the T/G-rC heteroduplex. (Left panels) MMR assay carried out in extracts of 293T cells immunodepleted of RNase H2. The figure shows an agarose gel image (GelRed) and autoradiograph ( 32 P) of 5 and 15 min time points of a representative experiment. (Top right) Plot of data from the 15 min time points of three independent experiments, with error bars representing standard deviation from the mean. (Bottom right) Immunodepletion of RNase H2 and supplementation of 293T extracts with recombinant MutLα. This western blot shows the efficiency of the immunodepletion procedure and the amounts of recombinant MLH1 relative to endogenous MSH2 levels. (C) Extracts of RNase H2 knockout mouse embryonal fibroblasts display decreased MMR activity on the T/G-rC heteroduplex at the 15 and 30 min time points. (Left panel) An agarose gel image (GelRed) and autoradiograph ( 32 P) of a representative experiment. (Top right) Plot of data from the 30 min time points of three independent experiments, with error bars representing standard deviation from the mean. (Bottom right) Western blot showing the amount of RNASEH2A in the MEFs as well as the amount of recombinant RNase H2 protein added to restore the MMR defect. MSH2 visualized with an anti-MSH2 antibody served as the loading control in the above experiments. M, size marker (1 kb ladder, New England BioLabs).

Techniques Used: Activity Assay, Transfection, Agarose Gel Electrophoresis, Autoradiography, Standard Deviation, Western Blot, Luciferase, Recombinant, Knock-Out, Marker

29) Product Images from "Differential expression of apoptotic genes PDIA3 and MAP3K5 distinguishes between low- and high-risk prostate cancer"

Article Title: Differential expression of apoptotic genes PDIA3 and MAP3K5 distinguishes between low- and high-risk prostate cancer

Journal: Molecular Cancer

doi: 10.1186/1476-4598-8-130

siRNA mediated knockdown of PDIA3 decreased apoptosis in prostate cancer cell line . (A) Knockdown efficiency measured via qRT PCR 48 h after transfection with 20 nM siRNA. (B) PC3 cells were treated with 20 nM scrambled siRNA control and PDIA3 siRNA. 48 h after transfection induction of apoptosis was performed with 1 μM Staurosporine (STS), 20 μM Fenretinide (FenR) or 1.5 μM Tapsigargin (TG) for 6 and 24 hours. Apoptosis was measured by determining caspase activation and compared to untreated control. Bar heights and error bars are means and upper range of triplicate samples relative to control treatment. * P
Figure Legend Snippet: siRNA mediated knockdown of PDIA3 decreased apoptosis in prostate cancer cell line . (A) Knockdown efficiency measured via qRT PCR 48 h after transfection with 20 nM siRNA. (B) PC3 cells were treated with 20 nM scrambled siRNA control and PDIA3 siRNA. 48 h after transfection induction of apoptosis was performed with 1 μM Staurosporine (STS), 20 μM Fenretinide (FenR) or 1.5 μM Tapsigargin (TG) for 6 and 24 hours. Apoptosis was measured by determining caspase activation and compared to untreated control. Bar heights and error bars are means and upper range of triplicate samples relative to control treatment. * P

Techniques Used: Quantitative RT-PCR, Transfection, Activation Assay

30) Product Images from "Biogenesis of mammalian microRNAs by a non-canonical processing pathway"

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks026

Simtron biogenesis involves Drosha but not DGCR8. Knockdown of DGCR8 in HeLa cells using siRNA was quantitated by ( A ) RT–PCR analysis of DGCR8 mRNA and ( B ) western blot analysis of DGCR8 protein expression. The percentage of knockdown of DGCR8 was quantitated for DGCR8 mRNA using the equation 100 − [(( DGCR8 knockdown / GAPDH )/( DGCR8 control / GAPDH )) × 100], n = 5 and for DGCR8 protein using the equation 100 − [(( DGCR8 knockdown /β-actin )/( DGCR8 control / β-actin )) × 100]. (C) Changes in endogenous miRNA levels following DGCR8 knockdown were analysed by stemloop RT–PCR analysis. miR-16 is a canonical miRNA control and sno65 is a loading control. Graph shows quantitation of miRNA abundance using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). n = 4 for all miRNAs except for miR-16, n = 5; asterisk indicates P ≤ 0.05 (Wilcoxon matched pairs signed-rank test). M indicates a synthetic size marker and filled circle indicates a non-specific primer dimer. ( D ) RT–PCR analysis of Drosha mRNA following expression of TN-Drosha in HEK-293T cells. ( E ) The effect of TN-Drosha expression on endogenous miRNA abundance was analysed by stemloop RT–PCR. Graph shows quantitation of miRNA abundance using the same equation as in C, n = 6; asterick indicates P ≤ 0.05 (Student's t -test). ( F ) Stemloop RT–PCR analysis of minigene-derived miR-877, 1226, 1225, 1228 and endogenous miR-16 isolated from HEK-293T cells transiently transfected with TN-Drosha. sno65 was used as a control. TN-Drosha mRNA expression in HEK-293T cells was analysed by radiolabelled RT–PCR. GAPDH was used as a control. ( G ) Quantitation of miRNA abundance relative to sno65 using the equation: miRNA/sno65. n = 3 for miR-877, 1226 and 1225, n = 5 for miR-1228 and n = 14 for miR-16; * P ≤ 0.05, *** P ≤ 0.0001 (). Data sets were analysed using the Student's t -test with the exception of miR-16, which was analysed using the Wilcoxon matched pairs signed-rank test. In all panels, bars represent the average ± SEM. The horizontal dotted line indicates normalized control levels.
Figure Legend Snippet: Simtron biogenesis involves Drosha but not DGCR8. Knockdown of DGCR8 in HeLa cells using siRNA was quantitated by ( A ) RT–PCR analysis of DGCR8 mRNA and ( B ) western blot analysis of DGCR8 protein expression. The percentage of knockdown of DGCR8 was quantitated for DGCR8 mRNA using the equation 100 − [(( DGCR8 knockdown / GAPDH )/( DGCR8 control / GAPDH )) × 100], n = 5 and for DGCR8 protein using the equation 100 − [(( DGCR8 knockdown /β-actin )/( DGCR8 control / β-actin )) × 100]. (C) Changes in endogenous miRNA levels following DGCR8 knockdown were analysed by stemloop RT–PCR analysis. miR-16 is a canonical miRNA control and sno65 is a loading control. Graph shows quantitation of miRNA abundance using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). n = 4 for all miRNAs except for miR-16, n = 5; asterisk indicates P ≤ 0.05 (Wilcoxon matched pairs signed-rank test). M indicates a synthetic size marker and filled circle indicates a non-specific primer dimer. ( D ) RT–PCR analysis of Drosha mRNA following expression of TN-Drosha in HEK-293T cells. ( E ) The effect of TN-Drosha expression on endogenous miRNA abundance was analysed by stemloop RT–PCR. Graph shows quantitation of miRNA abundance using the same equation as in C, n = 6; asterick indicates P ≤ 0.05 (Student's t -test). ( F ) Stemloop RT–PCR analysis of minigene-derived miR-877, 1226, 1225, 1228 and endogenous miR-16 isolated from HEK-293T cells transiently transfected with TN-Drosha. sno65 was used as a control. TN-Drosha mRNA expression in HEK-293T cells was analysed by radiolabelled RT–PCR. GAPDH was used as a control. ( G ) Quantitation of miRNA abundance relative to sno65 using the equation: miRNA/sno65. n = 3 for miR-877, 1226 and 1225, n = 5 for miR-1228 and n = 14 for miR-16; * P ≤ 0.05, *** P ≤ 0.0001 (). Data sets were analysed using the Student's t -test with the exception of miR-16, which was analysed using the Wilcoxon matched pairs signed-rank test. In all panels, bars represent the average ± SEM. The horizontal dotted line indicates normalized control levels.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Quantitation Assay, Marker, Derivative Assay, Isolation, Transfection

Simtron biogenesis does not require DGCR8, Dicer, Ago2 or XPO5. ( A ) RT–PCR analysis of minigene-derived host gene mRNA and stemloop RT–PCR analysis of minigene-derived miRNA and endogenous miR-16 in Dicer and DGCR8 knockout mouse embryonic stem cells transfected with the wt or splicing-deficient minigene (Δss) or empty vector control (−). sno65 was used as a loading control. Graphs show quantitation of miRNA using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). Bars represent the average ± SEM, n = 3. The horizontal dotted lines indicate normalized control levels. ( B ) Stemloop RT–PCR analysis of miR-1225 and miR-1228 immunoprecipitated from HEK-293T cell lysates that were transiently transfected with wt or Δss minigenes, or miR-877 from wt minigene along with pFLAG-Dicer (Dicer) or without (−) and immunoprecipitated with an antibody against the FLAG epitope. Input refers to cell lysates before FLAG immunoprecipitation; Un is the unbound fraction and IP is the immunoprecipitated fraction. Un is 1/20 IP and Input is 1/5 IP. The graph represents the percent of the mature miRNA found in the IP fraction versus the amount that remained in the Un fraction using the equation: (IP/(IP + (Un × 20)) × 100). ( C ) Stemloop RT–PCR analysis of minigene-derived miR-1225, miR-1228 and endogenous miR-16 from Ago2 knockout mouse embryonic fibroblasts. sno65 was used as a loading control. Cells were transiently transfected with wt or Δss minigenes or empty vector control (−). Graph shows quantitation of miRNA abundance using the same equation as in A. Bars represent the average ± SEM, n = 3 and * P ≤ 0.05 or ** P ≤ 0.01, Student's t -test. The horizontal dotted lines indicate normalized control levels. ( D ) Stemloop RT–PCR and RT–PCR analysis of miR-877 (left panel), miR-1225 (middle panel) and miR-1228 (right panel) minigene-expression in HeLa cells following siRNA-directed knockdown of XPO5 . sno65 is a loading control for miRNA using stemloop RT–PCR and GAPDH is a loading control for RT–PCR of XPO5 mRNA.
Figure Legend Snippet: Simtron biogenesis does not require DGCR8, Dicer, Ago2 or XPO5. ( A ) RT–PCR analysis of minigene-derived host gene mRNA and stemloop RT–PCR analysis of minigene-derived miRNA and endogenous miR-16 in Dicer and DGCR8 knockout mouse embryonic stem cells transfected with the wt or splicing-deficient minigene (Δss) or empty vector control (−). sno65 was used as a loading control. Graphs show quantitation of miRNA using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). Bars represent the average ± SEM, n = 3. The horizontal dotted lines indicate normalized control levels. ( B ) Stemloop RT–PCR analysis of miR-1225 and miR-1228 immunoprecipitated from HEK-293T cell lysates that were transiently transfected with wt or Δss minigenes, or miR-877 from wt minigene along with pFLAG-Dicer (Dicer) or without (−) and immunoprecipitated with an antibody against the FLAG epitope. Input refers to cell lysates before FLAG immunoprecipitation; Un is the unbound fraction and IP is the immunoprecipitated fraction. Un is 1/20 IP and Input is 1/5 IP. The graph represents the percent of the mature miRNA found in the IP fraction versus the amount that remained in the Un fraction using the equation: (IP/(IP + (Un × 20)) × 100). ( C ) Stemloop RT–PCR analysis of minigene-derived miR-1225, miR-1228 and endogenous miR-16 from Ago2 knockout mouse embryonic fibroblasts. sno65 was used as a loading control. Cells were transiently transfected with wt or Δss minigenes or empty vector control (−). Graph shows quantitation of miRNA abundance using the same equation as in A. Bars represent the average ± SEM, n = 3 and * P ≤ 0.05 or ** P ≤ 0.01, Student's t -test. The horizontal dotted lines indicate normalized control levels. ( D ) Stemloop RT–PCR and RT–PCR analysis of miR-877 (left panel), miR-1225 (middle panel) and miR-1228 (right panel) minigene-expression in HeLa cells following siRNA-directed knockdown of XPO5 . sno65 is a loading control for miRNA using stemloop RT–PCR and GAPDH is a loading control for RT–PCR of XPO5 mRNA.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Knock-Out, Transfection, Plasmid Preparation, Quantitation Assay, Immunoprecipitation, FLAG-tag, Expressing

31) Product Images from "Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention"

Article Title: Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention

Journal: Scientific Reports

doi: 10.1038/srep02623

Ubb -KD was more cytotoxic to MCF7 than MCF10A cells. (a) MCF7 and MCF10A cells were transfected with control siRNA or Ubb siRNA (20 nM) for 72 h and then observed by light microscopy. The levels of Ub in the transfected cells were compared by Western blotting with an anti-Ub antibody; α−tubulin was used as a control. (b) Relative cell proliferation was measured by MTT assay (n = 5). (c) The proportion of apoptotic cells at 72 h was measured by FACS (n = 2). The amount of cleaved PARP was also analyzed by Western blot. (d) DNA fragmentation was assessed from isolated genomic DNA. The data are shown as the mean ± SD, ***, p
Figure Legend Snippet: Ubb -KD was more cytotoxic to MCF7 than MCF10A cells. (a) MCF7 and MCF10A cells were transfected with control siRNA or Ubb siRNA (20 nM) for 72 h and then observed by light microscopy. The levels of Ub in the transfected cells were compared by Western blotting with an anti-Ub antibody; α−tubulin was used as a control. (b) Relative cell proliferation was measured by MTT assay (n = 5). (c) The proportion of apoptotic cells at 72 h was measured by FACS (n = 2). The amount of cleaved PARP was also analyzed by Western blot. (d) DNA fragmentation was assessed from isolated genomic DNA. The data are shown as the mean ± SD, ***, p

Techniques Used: Transfection, Light Microscopy, Western Blot, MTT Assay, FACS, Isolation

Ubb -KD inhibits cell proliferation and induces apoptotic cell death. (a) SH-SY5Y cells, PC3 cells, and HepG2 cells were transfected with 20 nM control siRNA or Ubb siRNA for 72 h and observed by light microscopy. The levels of Ub in siRNA-transfected cells were compared by Western blot. β-actin was used as a control. (b) Relative cell proliferation of siRNA-transfected cells was measured by MTT assay (n = 9). (c) Apoptotic cells at 72 h were assessed by measuring the sub-G1 population by FACS analysis (n = 2). Cleaved PARP cleavage was monitored as an apoptotic product by Western blot. Tubulin was used as a control. (d) The cell cycle distribution of the siRNA-transfected cells was analyzed at the indicated time by FACS. The 2 values in each panel indicate the percentage of cells at sub-G1 and G2/M stages. The data are shown as the mean ± SD, **, p
Figure Legend Snippet: Ubb -KD inhibits cell proliferation and induces apoptotic cell death. (a) SH-SY5Y cells, PC3 cells, and HepG2 cells were transfected with 20 nM control siRNA or Ubb siRNA for 72 h and observed by light microscopy. The levels of Ub in siRNA-transfected cells were compared by Western blot. β-actin was used as a control. (b) Relative cell proliferation of siRNA-transfected cells was measured by MTT assay (n = 9). (c) Apoptotic cells at 72 h were assessed by measuring the sub-G1 population by FACS analysis (n = 2). Cleaved PARP cleavage was monitored as an apoptotic product by Western blot. Tubulin was used as a control. (d) The cell cycle distribution of the siRNA-transfected cells was analyzed at the indicated time by FACS. The 2 values in each panel indicate the percentage of cells at sub-G1 and G2/M stages. The data are shown as the mean ± SD, **, p

Techniques Used: Transfection, Light Microscopy, Western Blot, MTT Assay, FACS

Ubb -KD stabilizes ubiquitination-dependent substrates and induces the expression of stress proteins. (a) SH-SY5Y cells stably expressing GFPu were transfected with 20 nM control or Ubb siRNA for 48 h and then the level of GFPu was assessed by Western blot using anti-GFP antibody. β-actin was used as a control. (b) The levels of p53, ODC, and β-actin were compared in HepG2 cells transfected with 20 nM control or Ubb siRNA for 48 h. (c) HeLa cells stably expressing αTCR were transfected with 20 nM control or Ubb siRNA for 72 h, and the level of αTCR was compared by Western blotting with an anti-HA antibody. MG132 (10 μM) was treated for 24 h as positive control in (a), (b), and (c). (d) HeLa cells were transfected with control siRNA or Ubb siRNA (20 nM) for 48 h and then treated with EGF (100 ng/ml). The cells were harvested at the indicated times (0 h, 0.5 h, 1 h, 2 h, and 3 h), and the level of EGFR was compared by Western blot. (e) Graph showing optical densities of EGFR from the immunoblot in (d). Values were normalized by β-actin. (f) HeLa cells were transfected with control siRNA or Ubb siRNA (20 nM) for 72 h, and the levels of HSP70, GRP78, and β-actin were compared by Western blot. (g and h) Total RNA was isolated from siRNA-transfected HeLa cells after 48 h, and the levels of HSP70 and GRP78 mRNA were compared by PCR (g) and real-time PCR, values were normalized by β-actin mRNA (h). The data are shown as the mean ± SD (n = 2), *, p
Figure Legend Snippet: Ubb -KD stabilizes ubiquitination-dependent substrates and induces the expression of stress proteins. (a) SH-SY5Y cells stably expressing GFPu were transfected with 20 nM control or Ubb siRNA for 48 h and then the level of GFPu was assessed by Western blot using anti-GFP antibody. β-actin was used as a control. (b) The levels of p53, ODC, and β-actin were compared in HepG2 cells transfected with 20 nM control or Ubb siRNA for 48 h. (c) HeLa cells stably expressing αTCR were transfected with 20 nM control or Ubb siRNA for 72 h, and the level of αTCR was compared by Western blotting with an anti-HA antibody. MG132 (10 μM) was treated for 24 h as positive control in (a), (b), and (c). (d) HeLa cells were transfected with control siRNA or Ubb siRNA (20 nM) for 48 h and then treated with EGF (100 ng/ml). The cells were harvested at the indicated times (0 h, 0.5 h, 1 h, 2 h, and 3 h), and the level of EGFR was compared by Western blot. (e) Graph showing optical densities of EGFR from the immunoblot in (d). Values were normalized by β-actin. (f) HeLa cells were transfected with control siRNA or Ubb siRNA (20 nM) for 72 h, and the levels of HSP70, GRP78, and β-actin were compared by Western blot. (g and h) Total RNA was isolated from siRNA-transfected HeLa cells after 48 h, and the levels of HSP70 and GRP78 mRNA were compared by PCR (g) and real-time PCR, values were normalized by β-actin mRNA (h). The data are shown as the mean ± SD (n = 2), *, p

Techniques Used: Expressing, Stable Transfection, Transfection, Western Blot, Positive Control, Isolation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Ubb -KD inhibits tumor cell growth in vivo . (a) PC3 cells (1 × 10 7 ) transfected with either control siRNA or Ubb siRNA (8 nM each) were subcutaneously injected into the left and right flanks of nude mice (n = 11). The size of the tumors was measured every 5 days after injection. (b) The size of the tumors at day 40 after injection was compared. (c) The weight of the tumors was measured after dissection (n = 11). The data are shown as the mean ± SD, *, p
Figure Legend Snippet: Ubb -KD inhibits tumor cell growth in vivo . (a) PC3 cells (1 × 10 7 ) transfected with either control siRNA or Ubb siRNA (8 nM each) were subcutaneously injected into the left and right flanks of nude mice (n = 11). The size of the tumors was measured every 5 days after injection. (b) The size of the tumors at day 40 after injection was compared. (c) The weight of the tumors was measured after dissection (n = 11). The data are shown as the mean ± SD, *, p

Techniques Used: In Vivo, Transfection, Injection, Mouse Assay, Dissection

Ubb -KD inhibits TNFα-mediated NF-κB activation. (a) HeLa cells were co-transfected with an NF-κB(2x) luciferase plasmid (30 ng), HSP70-β–galactosidase plasmid (30 ng), and control siRNA or Ubb siRNA (20 nM) for 48 h and then stimulated with 50 ng/ml TNFα for 2 h. Cells were harvested, and luciferase and β-galactosidase activity was measured. Luciferase activity was normalized to β-galactosidase activity. The data are shown as the mean ± SD (n = 6), ***, p
Figure Legend Snippet: Ubb -KD inhibits TNFα-mediated NF-κB activation. (a) HeLa cells were co-transfected with an NF-κB(2x) luciferase plasmid (30 ng), HSP70-β–galactosidase plasmid (30 ng), and control siRNA or Ubb siRNA (20 nM) for 48 h and then stimulated with 50 ng/ml TNFα for 2 h. Cells were harvested, and luciferase and β-galactosidase activity was measured. Luciferase activity was normalized to β-galactosidase activity. The data are shown as the mean ± SD (n = 6), ***, p

Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Ub levels are downregulated by the knockdown of Ubb mRNA with siRNA. (a) Schematic representation of the 4 Ub genes; Rps27a and Uba52 encode Ub as a fusion protein with the ribosomal subunits S27a and L40, respectively, whereas Ubb and Ubc encode tandem units of Ub with 3 and 9 repeats, respectively. All Ub gene products are known to be processed co-translationally. (b) SH-SY5Y neuroblastoma cells were transfected with 20 nM control siRNA or Ubb siRNA for 48 h, and the mRNA level of each Ub gene was analyzed by PCR. Ubb siRNA effectively and specifically depleted Ubb mRNA. β-actin mRNA was used as a control. (c) SH-SY5Y cells were transfected with different concentrations of Ubb siRNA (5, 10, and 20 nM) for 48 h, and the Ub level was analyzed by Western blotting following SDS-PAGE of whole protein extracts. A mono-Ub (mUb) can be resolved from conjugated Ubs appeared as smear and several discrete bands including ubiquitinated histone (arrow). (d) The amounts of mono-Ub and conjugated Ub were independently compared by densitometry (n = 2). Both decreased in a dose-dependent manner, but the downregulation of mono-Ub was particularly noticeable. The data are shown as the mean ± SD, #, p
Figure Legend Snippet: Ub levels are downregulated by the knockdown of Ubb mRNA with siRNA. (a) Schematic representation of the 4 Ub genes; Rps27a and Uba52 encode Ub as a fusion protein with the ribosomal subunits S27a and L40, respectively, whereas Ubb and Ubc encode tandem units of Ub with 3 and 9 repeats, respectively. All Ub gene products are known to be processed co-translationally. (b) SH-SY5Y neuroblastoma cells were transfected with 20 nM control siRNA or Ubb siRNA for 48 h, and the mRNA level of each Ub gene was analyzed by PCR. Ubb siRNA effectively and specifically depleted Ubb mRNA. β-actin mRNA was used as a control. (c) SH-SY5Y cells were transfected with different concentrations of Ubb siRNA (5, 10, and 20 nM) for 48 h, and the Ub level was analyzed by Western blotting following SDS-PAGE of whole protein extracts. A mono-Ub (mUb) can be resolved from conjugated Ubs appeared as smear and several discrete bands including ubiquitinated histone (arrow). (d) The amounts of mono-Ub and conjugated Ub were independently compared by densitometry (n = 2). Both decreased in a dose-dependent manner, but the downregulation of mono-Ub was particularly noticeable. The data are shown as the mean ± SD, #, p

Techniques Used: Transfection, Polymerase Chain Reaction, Western Blot, SDS Page

32) Product Images from "Apoptosis resistance downstream of eIF4E: posttranscriptional activation of an anti-apoptotic transcript carrying a consensus hairpin structure"

Article Title: Apoptosis resistance downstream of eIF4E: posttranscriptional activation of an anti-apoptotic transcript carrying a consensus hairpin structure

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl558

Validation of osteopontin (Spp1) as a translationally activated antagonist of apoptosis. ( A ) RT–PCR quantification of the osteopontin transcript across the polyribosome gradient. Adjacent polyribosome fractions were paired, and osteopontin transcript was quantified by RT–PCR. ( B ) Representative experiment showing osteopontin protein levels in NIH 3T3 and NIH 3T3/4E cells after 16 h serum starvation (upper panel); and osteopontin levels in NIH 3T3/4E cells 48 h after transfection with osteopontin siRNA#1-scrambled (SCR), osteopontin siRNA#1 (os1) and osteopontin siRNA#2 (os2) at 20 nM. ( C ) Osteopontin total mRNA levels in NIH 3T3 and NIH 3T3/4E cells in complete (serum +) and media lacking serum (serum −). (Error bars indicate standard deviation from two independent measurements.) ( D ) Apoptotic frequency of NIH 3T3/4E cells 48 h after serum withdrawal in response to osteopontin (Spp1) siRNA#1 or osteopontin (Spp1) siRNA#1-scrambled siRNA. (Error bars indicate standard deviation from two independent experiments.)
Figure Legend Snippet: Validation of osteopontin (Spp1) as a translationally activated antagonist of apoptosis. ( A ) RT–PCR quantification of the osteopontin transcript across the polyribosome gradient. Adjacent polyribosome fractions were paired, and osteopontin transcript was quantified by RT–PCR. ( B ) Representative experiment showing osteopontin protein levels in NIH 3T3 and NIH 3T3/4E cells after 16 h serum starvation (upper panel); and osteopontin levels in NIH 3T3/4E cells 48 h after transfection with osteopontin siRNA#1-scrambled (SCR), osteopontin siRNA#1 (os1) and osteopontin siRNA#2 (os2) at 20 nM. ( C ) Osteopontin total mRNA levels in NIH 3T3 and NIH 3T3/4E cells in complete (serum +) and media lacking serum (serum −). (Error bars indicate standard deviation from two independent measurements.) ( D ) Apoptotic frequency of NIH 3T3/4E cells 48 h after serum withdrawal in response to osteopontin (Spp1) siRNA#1 or osteopontin (Spp1) siRNA#1-scrambled siRNA. (Error bars indicate standard deviation from two independent experiments.)

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Standard Deviation

33) Product Images from "Whole-genome screening identifies proteins localized to distinct nuclear bodies"

Article Title: Whole-genome screening identifies proteins localized to distinct nuclear bodies

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201303145

Comparison of our study with datasets on nuclear subcompartments. Datasets were created for each nuclear subcompartment based on online databases or recent review articles. NOPdb ( Ahmad et al., 2009 ) was used to represent nucleolar proteins. A proteomic analysis of interchromatin granule clusters ( Saitoh et al., 2004 ) was used to represent the nuclear speckles dataset. A PML body interactome analysis ( Van Damme et al., 2010 ) was used to represent proteins in PML bodies. A list of Cajal body proteins from a recent review paper ( Machyna et al., 2013 ) was used to represent proteins in Cajal bodies. A list of paraspeckle proteins from a review ( Bond and Fox, 2009 ) was used to represent proteins in paraspeckles. Venn graphs were used to show the extent of overlapping between an available dataset (green) and our study (blue). A group of proteins that are uniquely identified in our study and have been reported by others in the literature were presented in dark blue. The other groups of proteins that are confirmed by our shRNA screen to be involved in the assembly of Cajal bodies or paraspeckles were presented in yellow. Please also see Table S2 and Table S3 .
Figure Legend Snippet: Comparison of our study with datasets on nuclear subcompartments. Datasets were created for each nuclear subcompartment based on online databases or recent review articles. NOPdb ( Ahmad et al., 2009 ) was used to represent nucleolar proteins. A proteomic analysis of interchromatin granule clusters ( Saitoh et al., 2004 ) was used to represent the nuclear speckles dataset. A PML body interactome analysis ( Van Damme et al., 2010 ) was used to represent proteins in PML bodies. A list of Cajal body proteins from a recent review paper ( Machyna et al., 2013 ) was used to represent proteins in Cajal bodies. A list of paraspeckle proteins from a review ( Bond and Fox, 2009 ) was used to represent proteins in paraspeckles. Venn graphs were used to show the extent of overlapping between an available dataset (green) and our study (blue). A group of proteins that are uniquely identified in our study and have been reported by others in the literature were presented in dark blue. The other groups of proteins that are confirmed by our shRNA screen to be involved in the assembly of Cajal bodies or paraspeckles were presented in yellow. Please also see Table S2 and Table S3 .

Techniques Used: shRNA

Identify proteins required for paraspeckle formation using shRNA screen. (A and B) Representative images showed colocalization of newly identified paraspeckle proteins with paraspeckles marker p54nrb (A) or NEAT1 long noncoding RNA (B). (C–E) Phenotypic screen for proteins affecting paraspeckles assembly. (C) A schematic flow for shRNA screen is presented. (D) Bar graph showed the percentage (±SD) of paraspeckles-containing cells determined by p54nrb or NEAT1 staining after the indicated shRNA treatment relative to control mock shRNA-treated cells ( n = 3 independent experiments). (E) Bar graph showed relative NEAT1 expression (±SD) to control mock shRNA-treated cells and normalized to GAPDH ( n = 3 independent experiments). Dotted lines display the level of control mock shRNA-treated cells for comparison. (F) Representative images showed phenotypes after shRNA transduction. The localization of paraspeckles foci was detected with the use of anti-p54nrb antibodies (top) or FITC-RNA probes against NEAT1 (bottom). Arrows show the paraspeckles foci labeled by anti-p54nrb antibodies (top) or RNA probes against NEAT1 (bottom). CTL, control. Bars, 10 µm.
Figure Legend Snippet: Identify proteins required for paraspeckle formation using shRNA screen. (A and B) Representative images showed colocalization of newly identified paraspeckle proteins with paraspeckles marker p54nrb (A) or NEAT1 long noncoding RNA (B). (C–E) Phenotypic screen for proteins affecting paraspeckles assembly. (C) A schematic flow for shRNA screen is presented. (D) Bar graph showed the percentage (±SD) of paraspeckles-containing cells determined by p54nrb or NEAT1 staining after the indicated shRNA treatment relative to control mock shRNA-treated cells ( n = 3 independent experiments). (E) Bar graph showed relative NEAT1 expression (±SD) to control mock shRNA-treated cells and normalized to GAPDH ( n = 3 independent experiments). Dotted lines display the level of control mock shRNA-treated cells for comparison. (F) Representative images showed phenotypes after shRNA transduction. The localization of paraspeckles foci was detected with the use of anti-p54nrb antibodies (top) or FITC-RNA probes against NEAT1 (bottom). Arrows show the paraspeckles foci labeled by anti-p54nrb antibodies (top) or RNA probes against NEAT1 (bottom). CTL, control. Bars, 10 µm.

Techniques Used: shRNA, Marker, Flow Cytometry, Staining, Expressing, Transduction, Labeling, CTL Assay

TOE1 is required for maintaining Cajal body integrity and efficient splicing. (A) TOE1 was down regulated in HeLa cells transfected by siRNA against TOE1 . (B) Knockdown of TOE1 affected the number and homogeneity of coilin foci. A control and two different siRNAs against TOE1 were used to knock down endogenous TOE1 expression in HeLa cells. Localization of TOE1 and coilin was detected by immunostaining using anti-TOE1 and anti-coilin. To recover the expression of TOE1, a construct encoding an siRNA-resistant form of TOE1 was cotransfected with si TOE1 -A. The exogenous protein was detected by the anti-Flag antibody. (C) The bar graph shows the percentage of cells (±SD) containing more than four coilin foci after the indicated treatment. WT, wild type. (D and E) Down-regulation of TOE1 disrupted localization of SMN complex and newly synthesized Sm-D1. Control siRNA (si CTL ) – or si TOE1 -A–treated cells were subjected to coimmunostaining using anti-coilin and anti-SMN antibodies (D) or anti-coilin and anti-Flag (for HA-Flag–tagged Sm-D1) antibodies (E). (F) Quantitative results showed the percentage of cells (±SD) in which the indicated proteins colocalized with coilin ( n = 3 independent experiments). (G) TOE1 is required for efficient splicing. A splicing reporter was introduced into HeLa cells or WI-38 cells with the indicated treatment. 24 h later, both spliced and unspliced RNAs were amplified from cDNA using the indicated primer sets. (H) The intensity of unspliced and spliced products was quantified by Quantity One software. The ratios of spliced to unspliced RNAs (±SD) were normalized by controls and presented as a bar graph for the indicated groups ( n = 3 independent experiments). (I) TOE1 down-regulation suppresses cell growth. Cells were harvested and counted at day 1–5 after siRNA transfection. The cell numbers (±SD) were plotted against the days after siRNA treatment ( n = 3 independent experiments). Bars, 10 µm.
Figure Legend Snippet: TOE1 is required for maintaining Cajal body integrity and efficient splicing. (A) TOE1 was down regulated in HeLa cells transfected by siRNA against TOE1 . (B) Knockdown of TOE1 affected the number and homogeneity of coilin foci. A control and two different siRNAs against TOE1 were used to knock down endogenous TOE1 expression in HeLa cells. Localization of TOE1 and coilin was detected by immunostaining using anti-TOE1 and anti-coilin. To recover the expression of TOE1, a construct encoding an siRNA-resistant form of TOE1 was cotransfected with si TOE1 -A. The exogenous protein was detected by the anti-Flag antibody. (C) The bar graph shows the percentage of cells (±SD) containing more than four coilin foci after the indicated treatment. WT, wild type. (D and E) Down-regulation of TOE1 disrupted localization of SMN complex and newly synthesized Sm-D1. Control siRNA (si CTL ) – or si TOE1 -A–treated cells were subjected to coimmunostaining using anti-coilin and anti-SMN antibodies (D) or anti-coilin and anti-Flag (for HA-Flag–tagged Sm-D1) antibodies (E). (F) Quantitative results showed the percentage of cells (±SD) in which the indicated proteins colocalized with coilin ( n = 3 independent experiments). (G) TOE1 is required for efficient splicing. A splicing reporter was introduced into HeLa cells or WI-38 cells with the indicated treatment. 24 h later, both spliced and unspliced RNAs were amplified from cDNA using the indicated primer sets. (H) The intensity of unspliced and spliced products was quantified by Quantity One software. The ratios of spliced to unspliced RNAs (±SD) were normalized by controls and presented as a bar graph for the indicated groups ( n = 3 independent experiments). (I) TOE1 down-regulation suppresses cell growth. Cells were harvested and counted at day 1–5 after siRNA transfection. The cell numbers (±SD) were plotted against the days after siRNA treatment ( n = 3 independent experiments). Bars, 10 µm.

Techniques Used: Transfection, Expressing, Immunostaining, Construct, Synthesized, CTL Assay, Amplification, Software

34) Product Images from "TLR dependent XBP-1 activation induces an autocrine loop in rheumatoid arthritis synoviocytes"

Article Title: TLR dependent XBP-1 activation induces an autocrine loop in rheumatoid arthritis synoviocytes

Journal: Journal of Autoimmunity

doi: 10.1016/j.jaut.2013.11.002

sXBP1 knock down and IL-6 expression following stimulation with SNAPIN. (A) qRT-PCR determining sXBP1 levels in RA SF after siRNA transfection, targeting either XBP1 (siXBP1) or a non-targeting control (siNT) for 48 h in total. Cells were treated with SNAPIN (5 μg/ml) for 24 h. (B) IL-6 levels in culture supernatants measured by ELISA after XBP1 knock down and SNAPIN treatment.
Figure Legend Snippet: sXBP1 knock down and IL-6 expression following stimulation with SNAPIN. (A) qRT-PCR determining sXBP1 levels in RA SF after siRNA transfection, targeting either XBP1 (siXBP1) or a non-targeting control (siNT) for 48 h in total. Cells were treated with SNAPIN (5 μg/ml) for 24 h. (B) IL-6 levels in culture supernatants measured by ELISA after XBP1 knock down and SNAPIN treatment.

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay

35) Product Images from "Human protein Staufen-2 promotes HIV-1 proliferation by positively regulating RNA export activity of viral protein Rev"

Article Title: Human protein Staufen-2 promotes HIV-1 proliferation by positively regulating RNA export activity of viral protein Rev

Journal: Retrovirology

doi: 10.1186/1742-4690-11-18

Effect of hStau-2 over-expression and knockdown on Rev export activity. A) Over-expression of hStau-2 increased RRE-RNA levels in the cytoplasmic fraction of HEK293T cells: hStau-2 over-expressed HEK293T cells or control cells were transfected with pNL4-3 pro-viral DNA. After 24 hrs of pNL4-3 transfection, total RNA was isolated from the cytoplasmic fractions and cDNA was prepared from 500 ng of RNA. Viral RRE was measured in the cytoplasmic fraction by absolute qRT-PCR. B) A leftward shift in CT curves for RRE-containing RNA upon hStau-2 overexpression. C) hStau-2 knockdown reduced RRE-RNA levels in the cytoplasmic fraction of HEK293T cells: hStau-2 siRNA or scrambled siRNA were transfected into HEK293T cells followed by pNL4-3 pro-viral DNA transfection. After 24 hrs of pNL4-3 transfection, total RNA was isolated from the cytoplasmic fractions and cDNA was prepared from 500 ng of RNA. Viral RRE was measured in the cytoplasmic fraction by absolute qRT-PCR. D) A rightward shift in CT curves for RRE-containing RNA upon hStau-2 knockdown. The experiments were done more than 3 times and the error bar represents ± SD. *p value ≤ 0.05 was taken as significant.
Figure Legend Snippet: Effect of hStau-2 over-expression and knockdown on Rev export activity. A) Over-expression of hStau-2 increased RRE-RNA levels in the cytoplasmic fraction of HEK293T cells: hStau-2 over-expressed HEK293T cells or control cells were transfected with pNL4-3 pro-viral DNA. After 24 hrs of pNL4-3 transfection, total RNA was isolated from the cytoplasmic fractions and cDNA was prepared from 500 ng of RNA. Viral RRE was measured in the cytoplasmic fraction by absolute qRT-PCR. B) A leftward shift in CT curves for RRE-containing RNA upon hStau-2 overexpression. C) hStau-2 knockdown reduced RRE-RNA levels in the cytoplasmic fraction of HEK293T cells: hStau-2 siRNA or scrambled siRNA were transfected into HEK293T cells followed by pNL4-3 pro-viral DNA transfection. After 24 hrs of pNL4-3 transfection, total RNA was isolated from the cytoplasmic fractions and cDNA was prepared from 500 ng of RNA. Viral RRE was measured in the cytoplasmic fraction by absolute qRT-PCR. D) A rightward shift in CT curves for RRE-containing RNA upon hStau-2 knockdown. The experiments were done more than 3 times and the error bar represents ± SD. *p value ≤ 0.05 was taken as significant.

Techniques Used: Over Expression, Activity Assay, Transfection, Isolation, Quantitative RT-PCR

Effect of hStau-2 expression on viral p24 levels. A) Over-expression of hStau-2 increased HIV-1 production: hStau-2 over-expressed HEK293T cells or control cells were transfected with pNL4-3 pro-viral DNA and scored after 48 hours for p24 by ELISA in the culture supernatant. The inset shows over-expression of hStau-2 after transfection with increasing concentration of hStau2-59 construct. There was a significant increase in the p24 levels when 0.5 and 1ug of hStau2-59 construct was used. B) The relative quantification of full length (9 kb) viral transcript. Viral transcripts were quantified from hStau-2 overexpressed and control cells by qRT-PCR and normalized to β-actin. C) siRNA mediated knockdown of hStau-2 reduced HIV-1 production: hStau-2 siRNA or scrambled siRNA were transfected one day prior to pNL4-3 transfection in HEK293T cells. Inset: Semiquantitative RT-PCR gel showing a decrease in hStau-2 expression when hStau-2 specific siRNA was used. The viral p24 levels in the culture supernatant were progressively reduced when hStau-2 specific siRNA was used in a dose dependent manner. The experiments were done more than 3 times and error bars represents ± SD. *p value ≤ 0.05 were taken as significant.
Figure Legend Snippet: Effect of hStau-2 expression on viral p24 levels. A) Over-expression of hStau-2 increased HIV-1 production: hStau-2 over-expressed HEK293T cells or control cells were transfected with pNL4-3 pro-viral DNA and scored after 48 hours for p24 by ELISA in the culture supernatant. The inset shows over-expression of hStau-2 after transfection with increasing concentration of hStau2-59 construct. There was a significant increase in the p24 levels when 0.5 and 1ug of hStau2-59 construct was used. B) The relative quantification of full length (9 kb) viral transcript. Viral transcripts were quantified from hStau-2 overexpressed and control cells by qRT-PCR and normalized to β-actin. C) siRNA mediated knockdown of hStau-2 reduced HIV-1 production: hStau-2 siRNA or scrambled siRNA were transfected one day prior to pNL4-3 transfection in HEK293T cells. Inset: Semiquantitative RT-PCR gel showing a decrease in hStau-2 expression when hStau-2 specific siRNA was used. The viral p24 levels in the culture supernatant were progressively reduced when hStau-2 specific siRNA was used in a dose dependent manner. The experiments were done more than 3 times and error bars represents ± SD. *p value ≤ 0.05 were taken as significant.

Techniques Used: Expressing, Over Expression, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Construct, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

36) Product Images from "NFAT5 Regulates HIV-1 in Primary Monocytes via a Highly Conserved Long Terminal Repeat Site"

Article Title: NFAT5 Regulates HIV-1 in Primary Monocytes via a Highly Conserved Long Terminal Repeat Site

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0020130

MDMs Constitutively Express NFAT5, Which Is Required for Replication of HIV-1 Representative Isolates from Subtypes B, C, and E (A) NFAT5 expression was detected in human MDMs using real-time PCR. NFAT5 mRNA expression was significantly inhibited 6 d after transfection with NFAT5 siRNA (NFAT5#1) compared with cells transfected with GFP siRNA or mock transfected ( p
Figure Legend Snippet: MDMs Constitutively Express NFAT5, Which Is Required for Replication of HIV-1 Representative Isolates from Subtypes B, C, and E (A) NFAT5 expression was detected in human MDMs using real-time PCR. NFAT5 mRNA expression was significantly inhibited 6 d after transfection with NFAT5 siRNA (NFAT5#1) compared with cells transfected with GFP siRNA or mock transfected ( p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection

37) Product Images from "Differential activation and function of Rho GTPases during Salmonella–host cell interactions"

Article Title: Differential activation and function of Rho GTPases during Salmonella–host cell interactions

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200605144

Independent activation of Cdc42 and Rac1 by Salmonella effector proteins in vivo. (A) COS-2 cells were transfected with plasmids expressing either Cdc42 N17 , short hairpin RNA directed to Cdc42, or a vector control (pSHAG). 2 d after transfection, cells were infected with either wild-type S. typhimurium (WT) or its isogenic invA mutant, which is defective for type III secretion and bacteria-induced signaling ( Galán et al., 1992 ). 20 min after infection, the relative levels of activated Rac1 were determined by the amount bound to GST-PAK-CRIB that was normalized to the amount of Rac1 in cell lysates analyzed by Western blotting. The intensity of the bands was quantified using ImageJ software. Equivalent results were obtained in several repetitions of this experiment. (B and C) Levels of Cdc42 or Rac in Henle-407 (B) or COS-2 (C) cells transfected with RNAi constructs. GTPase levels were analyzed in cells 2 d after transfection using RT-PCR (B) or Western blotting with antibodies directed to Cdc42, Rac1, or actin (C).
Figure Legend Snippet: Independent activation of Cdc42 and Rac1 by Salmonella effector proteins in vivo. (A) COS-2 cells were transfected with plasmids expressing either Cdc42 N17 , short hairpin RNA directed to Cdc42, or a vector control (pSHAG). 2 d after transfection, cells were infected with either wild-type S. typhimurium (WT) or its isogenic invA mutant, which is defective for type III secretion and bacteria-induced signaling ( Galán et al., 1992 ). 20 min after infection, the relative levels of activated Rac1 were determined by the amount bound to GST-PAK-CRIB that was normalized to the amount of Rac1 in cell lysates analyzed by Western blotting. The intensity of the bands was quantified using ImageJ software. Equivalent results were obtained in several repetitions of this experiment. (B and C) Levels of Cdc42 or Rac in Henle-407 (B) or COS-2 (C) cells transfected with RNAi constructs. GTPase levels were analyzed in cells 2 d after transfection using RT-PCR (B) or Western blotting with antibodies directed to Cdc42, Rac1, or actin (C).

Techniques Used: Activation Assay, In Vivo, Transfection, Expressing, shRNA, Plasmid Preparation, Infection, Mutagenesis, Western Blot, Software, Construct, Reverse Transcription Polymerase Chain Reaction

38) Product Images from "TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells"

Article Title: TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku484

Reduction of TCP1 proteins can decrease the antisense activity of PS-ASOs. ( A ) qRT-PCR analyses for the mRNA levels of three different TCP1 subunits in cells treated with siRNAs targeting the TCP1 subunits. UTC: mock-transfected control cells. ( B ) Western analyses for the protein levels of three TCP1 subunits in siRNA-treated cells. ACTB, α-tubulin and γ-tubulin were also detected. GAPDH served as a loading control. ( C ) The antisense activity of a PS-ASO targeting NCL1 mRNA was reduced in cells depleted of TCP1 subunits, as determined by qRT-PCR for the levels of NCL1 mRNA. ( D ) qRT-PCR for the level of Malat1 RNA in test cells transfected with a PS-ASO targeting Malat1 RNA, as in panel (C). In all panels, the error bars indicate standard deviation from three experiments.
Figure Legend Snippet: Reduction of TCP1 proteins can decrease the antisense activity of PS-ASOs. ( A ) qRT-PCR analyses for the mRNA levels of three different TCP1 subunits in cells treated with siRNAs targeting the TCP1 subunits. UTC: mock-transfected control cells. ( B ) Western analyses for the protein levels of three TCP1 subunits in siRNA-treated cells. ACTB, α-tubulin and γ-tubulin were also detected. GAPDH served as a loading control. ( C ) The antisense activity of a PS-ASO targeting NCL1 mRNA was reduced in cells depleted of TCP1 subunits, as determined by qRT-PCR for the levels of NCL1 mRNA. ( D ) qRT-PCR for the level of Malat1 RNA in test cells transfected with a PS-ASO targeting Malat1 RNA, as in panel (C). In all panels, the error bars indicate standard deviation from three experiments.

Techniques Used: Activity Assay, Quantitative RT-PCR, Transfection, Western Blot, Allele-specific Oligonucleotide, Standard Deviation

TCP1-β localizes in nuclear PS-bodies upon PS-ASO transfection. ( A ) Immunofluorescence staining of TCP1-β in control HeLa cells [(−)ASO] or HeLa cells transfected with 60-nM Cy3-labeled PS-ASO ISIS446654. The co-localization of TCP1-β (FITC) and PS-ASO in nuclear PS-bodies is indicated with white arrows. The scale bars: upper panels, 10 μm; lower panels, 5 μm. ( B ) Western analysis of TCP1-β protein in control cells transfected with (UTC+ASO) or without (UTC−ASO) 60-nM ASO ISIS116847 for 24 h, or in cells transfected with 5-nM TCP1-β siRNA for 24 h. TCP1-β protein was detected using antibody ab92756 (Abcam). γ-tubulin served as a loading control. ( C ) The PS-body localization of TCP1-β was not due to channel crosstalk. HeLa cells were transfected with 60 nM of FITC-labeled PS-ASO ISIS256903 and stained for TCP1-β (AF647) as described in panel (A). Scale bars, 5 μm. ( D ) Reduction in levels of TCP1-β did not completely block the formation of PS-bodies. HeLa cells treated with TCP1-β siRNA [(−)TCP1-β] for 24 h were transfected with 60-nM ISIS446654 for 5 h and stained for TCP1-β protein (FITC). UTC: untreated control cells. Scale bars: 20 μm.
Figure Legend Snippet: TCP1-β localizes in nuclear PS-bodies upon PS-ASO transfection. ( A ) Immunofluorescence staining of TCP1-β in control HeLa cells [(−)ASO] or HeLa cells transfected with 60-nM Cy3-labeled PS-ASO ISIS446654. The co-localization of TCP1-β (FITC) and PS-ASO in nuclear PS-bodies is indicated with white arrows. The scale bars: upper panels, 10 μm; lower panels, 5 μm. ( B ) Western analysis of TCP1-β protein in control cells transfected with (UTC+ASO) or without (UTC−ASO) 60-nM ASO ISIS116847 for 24 h, or in cells transfected with 5-nM TCP1-β siRNA for 24 h. TCP1-β protein was detected using antibody ab92756 (Abcam). γ-tubulin served as a loading control. ( C ) The PS-body localization of TCP1-β was not due to channel crosstalk. HeLa cells were transfected with 60 nM of FITC-labeled PS-ASO ISIS256903 and stained for TCP1-β (AF647) as described in panel (A). Scale bars, 5 μm. ( D ) Reduction in levels of TCP1-β did not completely block the formation of PS-bodies. HeLa cells treated with TCP1-β siRNA [(−)TCP1-β] for 24 h were transfected with 60-nM ISIS446654 for 5 h and stained for TCP1-β protein (FITC). UTC: untreated control cells. Scale bars: 20 μm.

Techniques Used: Allele-specific Oligonucleotide, Transfection, Immunofluorescence, Staining, Labeling, Western Blot, Blocking Assay

Reduction of RAN can lead to the formation of cytoplasmic PS-body-like structures. ( A ) Western analysis for RAN protein in cells treated or not with siRNA targeting RAN. Levels of γ-tubulin were detected and served as a loading control. ( B ) Reduction of RAN reduces the level of nuclear ASOs. Control HeLa cells (UTC) or cells treated with RAN siRNA [(−)RAN] were transfected with 50-nM Cy3-labeled ISIS446654 for 4 h, washed and images were taken of live cells. The PS-ASOs were pseudo-colored to white. The white arrows indicate the nuclear accumulation of PS-ASOs in control cells, whereas blue arrows indicate cells with reduced nuclear levels of PS-ASOs upon RAN reduction. Scale bar: 10 μm. ( C ) Reduction of RAN can cause formation of PS-body-like structures in the cytoplasm. Cells as used in panel (B) were fixed and stained for TCP1-β. White arrows indicate the cytoplasmic PS-body-like structures that contain PS-ASOs and TCP1-β protein. Scale bars: upper panels, 10 μm; lower panels, 5 μm. ( D ) TCP1-ϵ subunit does not localize in the cytoplasmic PS-body-like structures. RAN-depleted cells were transfected with PS-ASOs and co-stained for TCP1-β and TCP1-ϵ subunits. The PS-body-like structures are indicated with arrows, whereas the corresponding positions are marked with dashed circles in the TCP1-ϵ panel. Scale bar: 10 μm.
Figure Legend Snippet: Reduction of RAN can lead to the formation of cytoplasmic PS-body-like structures. ( A ) Western analysis for RAN protein in cells treated or not with siRNA targeting RAN. Levels of γ-tubulin were detected and served as a loading control. ( B ) Reduction of RAN reduces the level of nuclear ASOs. Control HeLa cells (UTC) or cells treated with RAN siRNA [(−)RAN] were transfected with 50-nM Cy3-labeled ISIS446654 for 4 h, washed and images were taken of live cells. The PS-ASOs were pseudo-colored to white. The white arrows indicate the nuclear accumulation of PS-ASOs in control cells, whereas blue arrows indicate cells with reduced nuclear levels of PS-ASOs upon RAN reduction. Scale bar: 10 μm. ( C ) Reduction of RAN can cause formation of PS-body-like structures in the cytoplasm. Cells as used in panel (B) were fixed and stained for TCP1-β. White arrows indicate the cytoplasmic PS-body-like structures that contain PS-ASOs and TCP1-β protein. Scale bars: upper panels, 10 μm; lower panels, 5 μm. ( D ) TCP1-ϵ subunit does not localize in the cytoplasmic PS-body-like structures. RAN-depleted cells were transfected with PS-ASOs and co-stained for TCP1-β and TCP1-ϵ subunits. The PS-body-like structures are indicated with arrows, whereas the corresponding positions are marked with dashed circles in the TCP1-ϵ panel. Scale bar: 10 μm.

Techniques Used: Western Blot, Transfection, Labeling, Staining

39) Product Images from "Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway"

Article Title: Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004371

Knock down of RPS19 induces autophagy. ( A ) Western blot analysis of RPS19 expression in GFP-LC3 HEK cells transfected with siScr or si RPS19 and either untreated or treated with 100 nM rapamycin overnight ( B ). Densitometer analysis of the ratio of RPS19 to actin expression from Fig. 1A . ( C ) Western blot analysis of LC3 expression in GFP-LC3 HEK cells transfected with siScr or si RPS19 either untreated or treated with 100 nM rapamycin overnight. ( D ) Confocal analysis of GFP-LC3 HEK cells transfected with siScr or si RPS19 and either untreated or treated with 50 nM bafilomycin A for 4 hours. Size bars = 10 µM. ( E ) Quantification of the number of GFP-LC3 puncta per cell from Fig. 1D . At least 8 shots from 3 independent transfections are quantified. ( F ) Quantification of the percent of cells in Fig. 1D with cytoplasmic GFP-LC3. ( G ) Representative electron micrographs of GFP-LC3 HEK cells transfected with si RPS19 and immunogold labeled with LC3 antibodies. Size bar on left panel = 500 nM, on right panel = 200 nM. ( H ) Western blot analysis of LC3 expression in CD34 + cells either not infected (NI), infected with a scrambled control (shScr) or infected with a shRNA against RPS19 (sh RPS19 ).
Figure Legend Snippet: Knock down of RPS19 induces autophagy. ( A ) Western blot analysis of RPS19 expression in GFP-LC3 HEK cells transfected with siScr or si RPS19 and either untreated or treated with 100 nM rapamycin overnight ( B ). Densitometer analysis of the ratio of RPS19 to actin expression from Fig. 1A . ( C ) Western blot analysis of LC3 expression in GFP-LC3 HEK cells transfected with siScr or si RPS19 either untreated or treated with 100 nM rapamycin overnight. ( D ) Confocal analysis of GFP-LC3 HEK cells transfected with siScr or si RPS19 and either untreated or treated with 50 nM bafilomycin A for 4 hours. Size bars = 10 µM. ( E ) Quantification of the number of GFP-LC3 puncta per cell from Fig. 1D . At least 8 shots from 3 independent transfections are quantified. ( F ) Quantification of the percent of cells in Fig. 1D with cytoplasmic GFP-LC3. ( G ) Representative electron micrographs of GFP-LC3 HEK cells transfected with si RPS19 and immunogold labeled with LC3 antibodies. Size bar on left panel = 500 nM, on right panel = 200 nM. ( H ) Western blot analysis of LC3 expression in CD34 + cells either not infected (NI), infected with a scrambled control (shScr) or infected with a shRNA against RPS19 (sh RPS19 ).

Techniques Used: Western Blot, Expressing, Transfection, Labeling, Infection, shRNA

40) Product Images from "Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in breast cancer cells: role of B7-H1 as an anti-apoptotic molecule"

Article Title: Doxorubicin downregulates cell surface B7-H1 expression and upregulates its nuclear expression in breast cancer cells: role of B7-H1 as an anti-apoptotic molecule

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr2605

The effect of doxorubicin on B7-H1 and AKT expression and induction of apoptosis . A) Column chart of the FACS data showing the effect of siRNA-B7-H1 inhibition on the percentage of apoptosis induced after 48-hours treatment of MDA-MB-231 cells with doxorubicin (n = 6, **indicated P
Figure Legend Snippet: The effect of doxorubicin on B7-H1 and AKT expression and induction of apoptosis . A) Column chart of the FACS data showing the effect of siRNA-B7-H1 inhibition on the percentage of apoptosis induced after 48-hours treatment of MDA-MB-231 cells with doxorubicin (n = 6, **indicated P

Techniques Used: Expressing, FACS, Inhibition, Multiple Displacement Amplification

Related Articles

Transfection:

Article Title: ERBB2-modulated ATG4B and autophagic cell death in human ARPE19 during oxidative stress
Article Snippet: .. Real-time PCR ARPE-19 cells transfected with siRNA were used for the extraction of total RNA with TRIzol Reagent (Invitrogen, 15596–018). .. 1 μg RNA was converted to cDNA with Reverse Transcriptase (Invitrogen, 18064–014), and ToolsQuant II Fast RT kit (Tools, Taiwan, KRT-BA06) was used for cDNA synthesis.

Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP
Article Snippet: .. Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction. ..

Article Title: Targeting c-MET by Tivantinib through synergistic activation of JNK/c-jun pathway in cholangiocarcinoma
Article Snippet: .. HuCC-T1 and EGI-1 cell lines were transfected with siRNA using Lipofectamine® 2000 Transfection Reagent (Thermo Scientific, Rockford, IL, USA) as per the supplier’s protocol. .. Transfection efficiency was evaluated after 24, 48, and 72 h using western blotting.

Article Title: Decreased Expression of Hepatic Low-Density Lipoprotein Receptor–Related Protein 1 in Hypothyroidism: A Novel Mechanism of Atherogenic Dyslipidemia in Hypothyroidism
Article Snippet: .. Each siRNA was transfected into HepG2 cells using Lipofectamine 2000 (Invitrogen). .. We determined the siRNA silencing efficiency by the real-time polymerase chain reaction of target gene mRNA.

Article Title: Inositol Polyphosphate 4-phosphatase Type II Regulation of Androgen Receptor Activity
Article Snippet: .. siRNA transfections Noncoding control, INPP4B, PTEN, and FOXA1 siRNAs were transfected at 50–100 pmol siRNA per well in 6 well cell culture plate using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) as recommended by the manufacturer. ..

Labeling:

Article Title: A ribonucleoprotein octamer for targeted siRNA delivery
Article Snippet: .. The siRNA in the complex was labeled with FAM (Thermofisher, Waltham, MA). .. Aliquots of 5 μL (10 pmol siRNA equivalent) was collected at various time points and probed on 1% agarose gel.

Infection:

Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP
Article Snippet: .. Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction. ..

Real-time Polymerase Chain Reaction:

Article Title: ERBB2-modulated ATG4B and autophagic cell death in human ARPE19 during oxidative stress
Article Snippet: .. Real-time PCR ARPE-19 cells transfected with siRNA were used for the extraction of total RNA with TRIzol Reagent (Invitrogen, 15596–018). .. 1 μg RNA was converted to cDNA with Reverse Transcriptase (Invitrogen, 18064–014), and ToolsQuant II Fast RT kit (Tools, Taiwan, KRT-BA06) was used for cDNA synthesis.

Cell Culture:

Article Title: Inositol Polyphosphate 4-phosphatase Type II Regulation of Androgen Receptor Activity
Article Snippet: .. siRNA transfections Noncoding control, INPP4B, PTEN, and FOXA1 siRNAs were transfected at 50–100 pmol siRNA per well in 6 well cell culture plate using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) as recommended by the manufacturer. ..

Sequencing:

Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma
Article Snippet: .. Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China). .. The following primer probe sets (Integrated DNA Technologies, Coralville, IA, USA) were used: survivin, forward: 5′-CAACCGGACGAATGCTTTT-3′; reverse: 5′-AAGAACTGGCCCTTCTTGGA-3′; probe: 5′-/5HEX/CCAGATGAC/ZEN/GACCCCATAGAGGAA/3IABkFQ/-3′; GAPDH, forward: 5′-AATCCCATCACCATCTTCCAG-3′; reverse: 5′-AAATGAGCCCCAGCCTTC-3′; probe: 5′-/5Cy5/CCAGCATCGCCCCACTTG ATTTT/3IAbRQSp/-3′; β-actin primers, forward: 5′-CATCGTGGGCCGCCCTAGGC-3′, reverse: 5′-GGGCCTCGGTGAGCAGCACA-3′ (Sangon Biotech, Shanghai, China).

Plasmid Preparation:

Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP
Article Snippet: .. Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher sirna
    Cellular uptake. Cellular uptake of <t>DP-CLPs–PTX–survivin</t> <t>siRNA</t> (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.
    Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna/product/Thermo Fisher
    Average 99 stars, based on 18683 article reviews
    Price from $9.99 to $1999.99
    sirna - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    89
    Thermo Fisher chop sirna
    Application of ATF6 <t>siRNA</t> promotes neuronal survival at 24 h after ICH. (A) Representative Western blot images. (B) Quantitative analyses of ATF6, <t>CHOP,</t> Bip, Bcl-2, Bax, cleaved caspase-3. n = 6 for each group. The bars represent the mean ± SD. ∗ p
    Chop Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chop sirna/product/Thermo Fisher
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    chop sirna - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    95
    Thermo Fisher sirnas
    Optineurin associates with Myo6 and releases melanosomal tubules. (A and B) SR-IFM on <t>MNT-1</t> cells costained for endogenous Myo6 and TYRP1 (A) and linear pixel values across n melanosomes (B; n = 130) show Myo6 (A; arrowheads, 4× boxed regions) localization to TYRP1 + melanosomal subdomain. (C and D) Percentage of Myo6/TYRP1 + melanosomes (C; n = 15 cells), and relative number of Myo6 + spots per n melanosome (D; n = 441). (E and F) SR-IFM of MNT-1 cells costained for endogenous optineurin and TYRP1 (E) and linear pixel values across n melanosomes (F; n = 95) show optineurin (E; arrowheads, 4× boxed regions) localization to TYRP1 + melanosomal subdomain. (G and H) Percentage of optineurin/TYRP1 + melanosomes (G; n = 20 cells) and relative number of optineurin + spots per n melanosome (H; n = 400). (I) SR-IFM on GFP-Myo6-CBD–expressing MNT-1 cells labeled for optineurin and TYRP1. Magnified views (4×) show Myo6 and optineurin codistribution on TYRP1 + melanosome subdomains. (J) Quantification of the percentage of melanosomes with overlapping GFP-Myo6-CBD and optineurin subdomains as in I ( n = 14 cells). (K) Western blot analysis of lysates and GFP IPs of GFP- or GFP-Myo6-CBD–expressing MNT-1 cells probed for optineurin or GFP antibodies. (L) Live imaging frames of MNT-1 cells expressing mCh-VAMP7 and treated with control or optineurin (Optn) <t>siRNAs.</t> Magnified areas (4×) of boxed regions in siOptn cells show tubules (arrowheads) associated with melanosomes (arrows). (M) Relative number of mCh-VAMP7 + melanosomal tubules during 40-s movies per 256-µm 2 area of cells treated as in L (siCtrl, n = 4 independent experiments; siOptn, n = 3 independent experiments). (N) Percentage of mCh-VAMP7 + tubules detaching from melanosomes in control- or optineurin-depleted cells (M and N; siCtrl, n = 4 independent experiments; siOptn, n = 3 independent experiments). Molecular masses are in kilodaltons. Data are presented as the mean ± SEM. Bars: (A, E, I, and L) 10 µm; (A, B, E, F, and L, magnifications) 1 µm; (I, magnifications) 500 nm. **, P
    Sirnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2569 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirnas/product/Thermo Fisher
    Average 95 stars, based on 2569 article reviews
    Price from $9.99 to $1999.99
    sirnas - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    Image Search Results


    Cellular uptake. Cellular uptake of DP-CLPs–PTX–survivin siRNA (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.

    Journal: Drug Delivery

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

    doi: 10.1080/10717544.2018.1494225

    Figure Lengend Snippet: Cellular uptake. Cellular uptake of DP-CLPs–PTX–survivin siRNA (A1–A5), DP-CLPs–scrambled siRNA (B1–B5), CLPs–PTX–survivin siRNA (C1–C5) or CLPs–scrambled siRNA (D1–D5) particles (lipid/siRNA weight ratio of 1:0.08) by U251-CD133+ cells (A1–D1 and A2–D2), U251-CD133– cells (A3–D3 and A4–D4) or BCECs (A5–D5 and A6–D6) was examined by fluorescence microscopy after 60 min incubation. Phase contrast images were obtained before each corresponding fluorescent image. Red: rhodamine. Scale bar: 100 μm.

    Article Snippet: Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China).

    Techniques: Fluorescence, Microscopy, Incubation

    Characterization of nanocomplex. (A) Agarose gel electrophoretic mobility shift assay was performed for DP-CLPs–PTX–survivin siRNA (M-e 1 ), DP-CLPs–scrambled siRNA (M-e 2 ), CLPs–PTX–survivin siRNA (M-e 3 ) and CLPs–scrambled siRNA (M-e 4 ). Control was in lane M. Lanes a, b, c, d and e corresponded to lipid/siRNA ratios of 1:0.05, 1:0.08, 1:0.1, 1:0.15 and 1:0.2 (w/w), respectively. (B) Atomic force microscopy pictures of (a) CLPs–scrambled siRNA, (b) DP-CLPs–scrambled siRNA, (c) CLPs–PTX–survivin siRNA siRNA and (d) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08. (C) PTX and DiR in vitro release profiles from the above liposomes. (D) TEM of (a) CLPs–PTX–survivin siRNA siRNA and (b) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08.

    Journal: Drug Delivery

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

    doi: 10.1080/10717544.2018.1494225

    Figure Lengend Snippet: Characterization of nanocomplex. (A) Agarose gel electrophoretic mobility shift assay was performed for DP-CLPs–PTX–survivin siRNA (M-e 1 ), DP-CLPs–scrambled siRNA (M-e 2 ), CLPs–PTX–survivin siRNA (M-e 3 ) and CLPs–scrambled siRNA (M-e 4 ). Control was in lane M. Lanes a, b, c, d and e corresponded to lipid/siRNA ratios of 1:0.05, 1:0.08, 1:0.1, 1:0.15 and 1:0.2 (w/w), respectively. (B) Atomic force microscopy pictures of (a) CLPs–scrambled siRNA, (b) DP-CLPs–scrambled siRNA, (c) CLPs–PTX–survivin siRNA siRNA and (d) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08. (C) PTX and DiR in vitro release profiles from the above liposomes. (D) TEM of (a) CLPs–PTX–survivin siRNA siRNA and (b) DP-CLPs–PTX–survivin siRNA siRNA at a lipid/siRNA weight ratio of 1:0.08.

    Article Snippet: Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China).

    Techniques: Agarose Gel Electrophoresis, Electrophoretic Mobility Shift Assay, Microscopy, In Vitro, Transmission Electron Microscopy

    (A) Quantification of the viability of U251-CD133 + cells, U251-CD133 – cells and BCECs after treatment with DMEM, CLPs–scrambled siRNA, DP-CLPs–scrambled siRNA, PTX, CLPs–PTX–survivin siRNA siRNA or DP-CLPs–PTX–survivin siRNA for 48 h. * p

    Journal: Drug Delivery

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

    doi: 10.1080/10717544.2018.1494225

    Figure Lengend Snippet: (A) Quantification of the viability of U251-CD133 + cells, U251-CD133 – cells and BCECs after treatment with DMEM, CLPs–scrambled siRNA, DP-CLPs–scrambled siRNA, PTX, CLPs–PTX–survivin siRNA siRNA or DP-CLPs–PTX–survivin siRNA for 48 h. * p

    Article Snippet: Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China).

    Techniques:

    (A) In vivo fluorescence imaging of intracranial U251-CD133 + glioma tumor-bearing nude mice treated for 24 h with CLPs–PTX–survivin siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ) liposomes, as well as corresponding dissected organs (A 1 and C 1 ). (B) Lesions in nude mice implanted in situ with U251-CD133 + cells after treatment with CLPs–scrambled siRNA (A 1 ), CLPs–PTX–survivin siRNA (C 1 ), DP-CLPs–scrambled siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ); all lesions were characterized by MRI at 19 days post-injection. U251-CD133 + cells were intracranially implanted in nude mice after 48 h treatment with Taxol, CLPs–PTX–survivin siRNA or DP-CLPs–PTX–survivin siRNA. Tumor size was measured 19 days post-injection ( n = 5/group). ** p

    Journal: Drug Delivery

    Article Title: Dual-modified cationic liposomes loaded with paclitaxel and survivin siRNA for targeted imaging and therapy of cancer stem cells in brain glioma

    doi: 10.1080/10717544.2018.1494225

    Figure Lengend Snippet: (A) In vivo fluorescence imaging of intracranial U251-CD133 + glioma tumor-bearing nude mice treated for 24 h with CLPs–PTX–survivin siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ) liposomes, as well as corresponding dissected organs (A 1 and C 1 ). (B) Lesions in nude mice implanted in situ with U251-CD133 + cells after treatment with CLPs–scrambled siRNA (A 1 ), CLPs–PTX–survivin siRNA (C 1 ), DP-CLPs–scrambled siRNA (B 1 ) or DP-CLPs–PTX–survivin siRNA (D 1 ); all lesions were characterized by MRI at 19 days post-injection. U251-CD133 + cells were intracranially implanted in nude mice after 48 h treatment with Taxol, CLPs–PTX–survivin siRNA or DP-CLPs–PTX–survivin siRNA. Tumor size was measured 19 days post-injection ( n = 5/group). ** p

    Article Snippet: Survivin siRNA (sequence: 5′-GCAUUCGUCCGGUUGCGCUdTdT-3′) and a scrambled siRNA (sequence: 5′-AUGAACUUCAGGGUCAGCUdTdT-3′) were purchased from Thermo Scientific Dharmacon (Shanghai, China).

    Techniques: In Vivo, Fluorescence, Imaging, Mouse Assay, In Situ, Magnetic Resonance Imaging, Injection

    Knockdown of CacyBP facilitated viral replication and arrested apoptosis in DF-1 cells. DF-1 cells were transfected with mock and/or NC and si-CacyBP/SIP and incubated for 36 h. Mock, treated with transfection regent; NC, transfected with si-NC; si183/326/644, separately transfected with those siRNAs targets CacyBP/SIP. (A) Endogenous CacyBP/SIP was detected by western blotting. (B) DF-1 cells were co-transfected with si-CacyBP (326) and pCMV-HA-CacyBP. Thirty-six hours after transfection, protein expression of HA-CacyBP was detected by anti-HA antibody through immunofluorescence in DF-1 cells. (C) Replication kinetics of NDV RNAs from the mock, NC, and siRNA (326) groups of DF-1 cells; 1 MOI of F48E9 NDV were inoculated into the three groups of cells 24 h after transfection. Q-PCR was used to measure viral RNA replication 24 hpi. (D) Viral plaque formation tests were further used to measure the number of viruses in the supernatants. (E) Three cell cultures were prepared and transfected with mock, NC and siRNA (326), and then, the cells were washed and harvested, an annexin V assay was followed by flow cytometry to monitor for percentage of cells undergoing early apoptosis (bottom right quadrant) and late apoptosis(upper right quadrant). Y-axis is PI signal; X-asis is annexin V-FITC signal. Right graph data were the percentage of total apoptosis (bottom and upper right quadrant)and data from three independent experiments. (F–H) Q-PCR was used to analyze the expression of apoptosis-related markers and immune-associated markers 24 h after transfection with NC and si-CacyBP/SIP. (G) The transfected cell lysate was analyzed by western blotting with the indicated apoptosis-related antibody. Data shown in (C,D) are mean ± SD of four independent experiments, in (E,G,H) are mean ± SD of three independent expriments. * P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP

    doi: 10.3389/fcimb.2018.00304

    Figure Lengend Snippet: Knockdown of CacyBP facilitated viral replication and arrested apoptosis in DF-1 cells. DF-1 cells were transfected with mock and/or NC and si-CacyBP/SIP and incubated for 36 h. Mock, treated with transfection regent; NC, transfected with si-NC; si183/326/644, separately transfected with those siRNAs targets CacyBP/SIP. (A) Endogenous CacyBP/SIP was detected by western blotting. (B) DF-1 cells were co-transfected with si-CacyBP (326) and pCMV-HA-CacyBP. Thirty-six hours after transfection, protein expression of HA-CacyBP was detected by anti-HA antibody through immunofluorescence in DF-1 cells. (C) Replication kinetics of NDV RNAs from the mock, NC, and siRNA (326) groups of DF-1 cells; 1 MOI of F48E9 NDV were inoculated into the three groups of cells 24 h after transfection. Q-PCR was used to measure viral RNA replication 24 hpi. (D) Viral plaque formation tests were further used to measure the number of viruses in the supernatants. (E) Three cell cultures were prepared and transfected with mock, NC and siRNA (326), and then, the cells were washed and harvested, an annexin V assay was followed by flow cytometry to monitor for percentage of cells undergoing early apoptosis (bottom right quadrant) and late apoptosis(upper right quadrant). Y-axis is PI signal; X-asis is annexin V-FITC signal. Right graph data were the percentage of total apoptosis (bottom and upper right quadrant)and data from three independent experiments. (F–H) Q-PCR was used to analyze the expression of apoptosis-related markers and immune-associated markers 24 h after transfection with NC and si-CacyBP/SIP. (G) The transfected cell lysate was analyzed by western blotting with the indicated apoptosis-related antibody. Data shown in (C,D) are mean ± SD of four independent experiments, in (E,G,H) are mean ± SD of three independent expriments. * P

    Article Snippet: Transfection and viral infection Plasmid or siRNA was transfected to DF-1 and/or Vero cells using Lipofectamine 2000 (Thermo Scientific, NH, USA) according to the manufacturer's instruction.

    Techniques: Transfection, Incubation, Western Blot, Expressing, Immunofluorescence, Polymerase Chain Reaction, Annexin V Assay, Flow Cytometry, Cytometry

    Application of ATF6 siRNA promotes neuronal survival at 24 h after ICH. (A) Representative Western blot images. (B) Quantitative analyses of ATF6, CHOP, Bip, Bcl-2, Bax, cleaved caspase-3. n = 6 for each group. The bars represent the mean ± SD. ∗ p

    Journal: Frontiers in Neuroscience

    Article Title: Melatonin Protects Against Neuronal Apoptosis via Suppression of the ATF6/CHOP Pathway in a Rat Model of Intracerebral Hemorrhage

    doi: 10.3389/fnins.2018.00638

    Figure Lengend Snippet: Application of ATF6 siRNA promotes neuronal survival at 24 h after ICH. (A) Representative Western blot images. (B) Quantitative analyses of ATF6, CHOP, Bip, Bcl-2, Bax, cleaved caspase-3. n = 6 for each group. The bars represent the mean ± SD. ∗ p

    Article Snippet: The results showed that the administration of ATF6 siRNA could significantly reduce the level of CHOP expression in both protein and mRNA levels, while CHOP siRNA had no effects on the expression of ATF6, which was elevated at 24 h after ICH (p < 0.05, vs. sham).

    Techniques: Western Blot

    Optineurin associates with Myo6 and releases melanosomal tubules. (A and B) SR-IFM on MNT-1 cells costained for endogenous Myo6 and TYRP1 (A) and linear pixel values across n melanosomes (B; n = 130) show Myo6 (A; arrowheads, 4× boxed regions) localization to TYRP1 + melanosomal subdomain. (C and D) Percentage of Myo6/TYRP1 + melanosomes (C; n = 15 cells), and relative number of Myo6 + spots per n melanosome (D; n = 441). (E and F) SR-IFM of MNT-1 cells costained for endogenous optineurin and TYRP1 (E) and linear pixel values across n melanosomes (F; n = 95) show optineurin (E; arrowheads, 4× boxed regions) localization to TYRP1 + melanosomal subdomain. (G and H) Percentage of optineurin/TYRP1 + melanosomes (G; n = 20 cells) and relative number of optineurin + spots per n melanosome (H; n = 400). (I) SR-IFM on GFP-Myo6-CBD–expressing MNT-1 cells labeled for optineurin and TYRP1. Magnified views (4×) show Myo6 and optineurin codistribution on TYRP1 + melanosome subdomains. (J) Quantification of the percentage of melanosomes with overlapping GFP-Myo6-CBD and optineurin subdomains as in I ( n = 14 cells). (K) Western blot analysis of lysates and GFP IPs of GFP- or GFP-Myo6-CBD–expressing MNT-1 cells probed for optineurin or GFP antibodies. (L) Live imaging frames of MNT-1 cells expressing mCh-VAMP7 and treated with control or optineurin (Optn) siRNAs. Magnified areas (4×) of boxed regions in siOptn cells show tubules (arrowheads) associated with melanosomes (arrows). (M) Relative number of mCh-VAMP7 + melanosomal tubules during 40-s movies per 256-µm 2 area of cells treated as in L (siCtrl, n = 4 independent experiments; siOptn, n = 3 independent experiments). (N) Percentage of mCh-VAMP7 + tubules detaching from melanosomes in control- or optineurin-depleted cells (M and N; siCtrl, n = 4 independent experiments; siOptn, n = 3 independent experiments). Molecular masses are in kilodaltons. Data are presented as the mean ± SEM. Bars: (A, E, I, and L) 10 µm; (A, B, E, F, and L, magnifications) 1 µm; (I, magnifications) 500 nm. **, P

    Journal: The Journal of Cell Biology

    Article Title: Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers

    doi: 10.1083/jcb.201709055

    Figure Lengend Snippet: Optineurin associates with Myo6 and releases melanosomal tubules. (A and B) SR-IFM on MNT-1 cells costained for endogenous Myo6 and TYRP1 (A) and linear pixel values across n melanosomes (B; n = 130) show Myo6 (A; arrowheads, 4× boxed regions) localization to TYRP1 + melanosomal subdomain. (C and D) Percentage of Myo6/TYRP1 + melanosomes (C; n = 15 cells), and relative number of Myo6 + spots per n melanosome (D; n = 441). (E and F) SR-IFM of MNT-1 cells costained for endogenous optineurin and TYRP1 (E) and linear pixel values across n melanosomes (F; n = 95) show optineurin (E; arrowheads, 4× boxed regions) localization to TYRP1 + melanosomal subdomain. (G and H) Percentage of optineurin/TYRP1 + melanosomes (G; n = 20 cells) and relative number of optineurin + spots per n melanosome (H; n = 400). (I) SR-IFM on GFP-Myo6-CBD–expressing MNT-1 cells labeled for optineurin and TYRP1. Magnified views (4×) show Myo6 and optineurin codistribution on TYRP1 + melanosome subdomains. (J) Quantification of the percentage of melanosomes with overlapping GFP-Myo6-CBD and optineurin subdomains as in I ( n = 14 cells). (K) Western blot analysis of lysates and GFP IPs of GFP- or GFP-Myo6-CBD–expressing MNT-1 cells probed for optineurin or GFP antibodies. (L) Live imaging frames of MNT-1 cells expressing mCh-VAMP7 and treated with control or optineurin (Optn) siRNAs. Magnified areas (4×) of boxed regions in siOptn cells show tubules (arrowheads) associated with melanosomes (arrows). (M) Relative number of mCh-VAMP7 + melanosomal tubules during 40-s movies per 256-µm 2 area of cells treated as in L (siCtrl, n = 4 independent experiments; siOptn, n = 3 independent experiments). (N) Percentage of mCh-VAMP7 + tubules detaching from melanosomes in control- or optineurin-depleted cells (M and N; siCtrl, n = 4 independent experiments; siOptn, n = 3 independent experiments). Molecular masses are in kilodaltons. Data are presented as the mean ± SEM. Bars: (A, E, I, and L) 10 µm; (A, B, E, F, and L, magnifications) 1 µm; (I, magnifications) 500 nm. **, P

    Article Snippet: For siRNAs, MNT-1 cells were seeded at day 1, transfected with siRNAs (200 pmol) using Oligofectamine (Thermo Fisher Scientific) at day 3, collected and analyzed at day 6 (for imaging experiments), or transfected again at day 5 and analyzed at day 8 (for biochemical analyses).

    Techniques: Expressing, Labeling, Western Blot, Imaging

    The WASH complex promotes melanosomal tubules constriction and fission. (A) Live imaging frames on mCh-VAMP7 expressing MNT-1 cells treated with control or WASH1 siRNAs. Magnified areas (4×) of boxed regions show tubules (arrowheads) associated with melanosomes (arrows). (B and C) Relative number of mCh-VAMP7 + melanosomal tubules (B) detaching from melanosomes (C) during 40-s acquisition per 256-µm 2 area of cells treated as in A (siCtrl, n = 4 independent experiments; siWASH1, n = 3 independent experiments). (D) High-pressure frozen control- or WASH1-depleted MNT-1 cells analyzed by 2D EM. Large tubules (arrowheads) emerge from melanosomes (arrows) upon siWASH1 treatment. (E) Percentage of melanosomes positive for tubule per n cell on EM section (siCtrl, n = 27; siWASH1, n = 21). (F and G) Mean length (F) of n tubules and width (G) of the neck (siCtrl, n = 41; siWASH1, n = 139). (H) Frequency of distribution of the width of tubules neck. Data are presented as the mean ± SEM. Bars: (A) 10 µm; (D and magnifications in A) 1 µm. ****, P

    Journal: The Journal of Cell Biology

    Article Title: Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers

    doi: 10.1083/jcb.201709055

    Figure Lengend Snippet: The WASH complex promotes melanosomal tubules constriction and fission. (A) Live imaging frames on mCh-VAMP7 expressing MNT-1 cells treated with control or WASH1 siRNAs. Magnified areas (4×) of boxed regions show tubules (arrowheads) associated with melanosomes (arrows). (B and C) Relative number of mCh-VAMP7 + melanosomal tubules (B) detaching from melanosomes (C) during 40-s acquisition per 256-µm 2 area of cells treated as in A (siCtrl, n = 4 independent experiments; siWASH1, n = 3 independent experiments). (D) High-pressure frozen control- or WASH1-depleted MNT-1 cells analyzed by 2D EM. Large tubules (arrowheads) emerge from melanosomes (arrows) upon siWASH1 treatment. (E) Percentage of melanosomes positive for tubule per n cell on EM section (siCtrl, n = 27; siWASH1, n = 21). (F and G) Mean length (F) of n tubules and width (G) of the neck (siCtrl, n = 41; siWASH1, n = 139). (H) Frequency of distribution of the width of tubules neck. Data are presented as the mean ± SEM. Bars: (A) 10 µm; (D and magnifications in A) 1 µm. ****, P

    Article Snippet: For siRNAs, MNT-1 cells were seeded at day 1, transfected with siRNAs (200 pmol) using Oligofectamine (Thermo Fisher Scientific) at day 3, collected and analyzed at day 6 (for imaging experiments), or transfected again at day 5 and analyzed at day 8 (for biochemical analyses).

    Techniques: Imaging, Expressing

    Melanin production, cargo export, melanosome secretion, and transfer rely on Myo6, WASH, or optineurin. (A) Western blot of total lysates of control-, Myo6-, or WASH1-depleted MNT-1 cells probed with respective antibodies. (B) Intracellular melanin estimation of control-, Myo6-, or WASH1-depleted MNT-1 cells ( n = 8 independent experiments; normalized to control). (C) Western blot of melanosome-enriched (Mel) fractions from control-, Myo6-, or WASH1-depleted MNT-1 cells probed with respective antibodies. (D) Quantification of TYRP1 and VAMP7 protein expression levels on Mel fractions in C and normalized to control ( n = 2 independent experiments). (E) GFP-VAMP7–expressing MNT-1 cells treated with siCtrl, siMyo6, or siWASH1 were processed for ultrathin cryosectioning and double immunogold labeled for GFP (PAG 10 nm, arrowheads) and TYRP1 (PAG 15 nm). (F and G) Percentage of GFP-VAMP7 + melanosome per n cell on section (F) and relative number of gold particles per GFP-VAMP7 + melanosome (G; siCtrl, n = 7; siMyo6, n = 6; siWASH1, n = 7). (H) Ratio of extracellular to intracellular melanin content of control-, Myo6-, or WASH1-depleted MNT-1 cells treated with 30 µM forskolin ( n = 3 independent experiments; normalized to control). (I) NHMs treated with control or Myo6 siRNAs were co-cultured with NHKs. Staining for HMB45 (red) or EGFR (green) identify melanin and NHM or NHK respectively. HMB45 staining within NHK (arrowheads) correspond to secreted and transferred melanin. (J and K) Quantification of the percentage of NHKs positive for at least one HMB45 + spot (J; n = 3 independent experiments) and of the mean HMB45 intensity per stained ( n ) NHK (K) when co-cultured with siCtrl, siMyo6, siWASH1 or siOptn NHM (siCtrl, n = 167; siMyo6, n = 147; siWASH1, n = 148; siOptn, n = 123). (L) Working model illustrating the roles of Myo6, optineurin, F-actin, WASH, Arp2/3, and BLOC-3 complexes in the recycling pathway from melanosomes. Molecular mass is in kilodaltons. Data are presented as the mean ± SEM. Bars: (E) 1 µm; (I) 10 µm. ****, P

    Journal: The Journal of Cell Biology

    Article Title: Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers

    doi: 10.1083/jcb.201709055

    Figure Lengend Snippet: Melanin production, cargo export, melanosome secretion, and transfer rely on Myo6, WASH, or optineurin. (A) Western blot of total lysates of control-, Myo6-, or WASH1-depleted MNT-1 cells probed with respective antibodies. (B) Intracellular melanin estimation of control-, Myo6-, or WASH1-depleted MNT-1 cells ( n = 8 independent experiments; normalized to control). (C) Western blot of melanosome-enriched (Mel) fractions from control-, Myo6-, or WASH1-depleted MNT-1 cells probed with respective antibodies. (D) Quantification of TYRP1 and VAMP7 protein expression levels on Mel fractions in C and normalized to control ( n = 2 independent experiments). (E) GFP-VAMP7–expressing MNT-1 cells treated with siCtrl, siMyo6, or siWASH1 were processed for ultrathin cryosectioning and double immunogold labeled for GFP (PAG 10 nm, arrowheads) and TYRP1 (PAG 15 nm). (F and G) Percentage of GFP-VAMP7 + melanosome per n cell on section (F) and relative number of gold particles per GFP-VAMP7 + melanosome (G; siCtrl, n = 7; siMyo6, n = 6; siWASH1, n = 7). (H) Ratio of extracellular to intracellular melanin content of control-, Myo6-, or WASH1-depleted MNT-1 cells treated with 30 µM forskolin ( n = 3 independent experiments; normalized to control). (I) NHMs treated with control or Myo6 siRNAs were co-cultured with NHKs. Staining for HMB45 (red) or EGFR (green) identify melanin and NHM or NHK respectively. HMB45 staining within NHK (arrowheads) correspond to secreted and transferred melanin. (J and K) Quantification of the percentage of NHKs positive for at least one HMB45 + spot (J; n = 3 independent experiments) and of the mean HMB45 intensity per stained ( n ) NHK (K) when co-cultured with siCtrl, siMyo6, siWASH1 or siOptn NHM (siCtrl, n = 167; siMyo6, n = 147; siWASH1, n = 148; siOptn, n = 123). (L) Working model illustrating the roles of Myo6, optineurin, F-actin, WASH, Arp2/3, and BLOC-3 complexes in the recycling pathway from melanosomes. Molecular mass is in kilodaltons. Data are presented as the mean ± SEM. Bars: (E) 1 µm; (I) 10 µm. ****, P

    Article Snippet: For siRNAs, MNT-1 cells were seeded at day 1, transfected with siRNAs (200 pmol) using Oligofectamine (Thermo Fisher Scientific) at day 3, collected and analyzed at day 6 (for imaging experiments), or transfected again at day 5 and analyzed at day 8 (for biochemical analyses).

    Techniques: Western Blot, Expressing, Labeling, Cell Culture, Staining