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    Thermo Fisher counterstain topro
    Immunohistochemistry for PrP Sc in heart in Syrian golden hamsters infected with chronic wasting disease. Skeletal muscle of heart from mock (D) and WST CWD-infected (A through C) hamsters. Panels A through C are the same field of view that are separated into three panels according to the immunofluorescence staining. Heart was analyzed by dual immunofluorescence for desmin (A, C, D) and PrP Sc (B, C, D). <t>ToPro®-3</t> staining of nuclei is indicated by blue fluorescence. Scale bar in A is 10 µm.
    Counterstain Topro, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 23669 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Transmission of Chronic Wasting Disease Identifies a Prion Strain Causing Cachexia and Heart Infection in Hamsters"

    Article Title: Transmission of Chronic Wasting Disease Identifies a Prion Strain Causing Cachexia and Heart Infection in Hamsters

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028026

    Immunohistochemistry for PrP Sc in heart in Syrian golden hamsters infected with chronic wasting disease. Skeletal muscle of heart from mock (D) and WST CWD-infected (A through C) hamsters. Panels A through C are the same field of view that are separated into three panels according to the immunofluorescence staining. Heart was analyzed by dual immunofluorescence for desmin (A, C, D) and PrP Sc (B, C, D). ToPro®-3 staining of nuclei is indicated by blue fluorescence. Scale bar in A is 10 µm.
    Figure Legend Snippet: Immunohistochemistry for PrP Sc in heart in Syrian golden hamsters infected with chronic wasting disease. Skeletal muscle of heart from mock (D) and WST CWD-infected (A through C) hamsters. Panels A through C are the same field of view that are separated into three panels according to the immunofluorescence staining. Heart was analyzed by dual immunofluorescence for desmin (A, C, D) and PrP Sc (B, C, D). ToPro®-3 staining of nuclei is indicated by blue fluorescence. Scale bar in A is 10 µm.

    Techniques Used: Immunohistochemistry, Infection, Immunofluorescence, Staining, Fluorescence

    Distribution of PrP Sc in olfactory sensory epithelium in Syrian golden hamsters infected with chronic wasting disease. Laser scanning confocal microscopy of olfactory marker protein (OMP)( A, G ), PrP Sc ( B, H ), and for both OMP and PrP Sc (Merge)( C, I ) in Syrian golden hamsters infected with TgMo-sghPrP CWD (A, B, C) and mock-infected hamsters (G, H, I). Laser scanning confocal microscopy of adenylyl cyclase III (ACIII)( D ), PrP Sc ( E ), and for both ACIII and PrP Sc (Merge)( F ) in CWD infected SGH. Panels A through C , D through F and G through I are the same field of view that are separated into three panels according to the immunofluorescence staining. ToPro®-3 staining of nuclei is indicated by blue fluorescence. Olfactory receptor neurons in the olfactory sensory epithelium (OSE width indicated by white line), and nerve fibers in the subepithelial layer (SE), both express high levels of OMP (A, C and G and I). ACIII is located on the sensory cilia that project from the terminal dendrites of ORNs and its distribution was prominent at the border between the OSE and airway lumen (D and F). The white arrow points to the distal edge of the OSE where it borders the lumen of the nasal airway. Scale bar in A, D and G is 50 µm.
    Figure Legend Snippet: Distribution of PrP Sc in olfactory sensory epithelium in Syrian golden hamsters infected with chronic wasting disease. Laser scanning confocal microscopy of olfactory marker protein (OMP)( A, G ), PrP Sc ( B, H ), and for both OMP and PrP Sc (Merge)( C, I ) in Syrian golden hamsters infected with TgMo-sghPrP CWD (A, B, C) and mock-infected hamsters (G, H, I). Laser scanning confocal microscopy of adenylyl cyclase III (ACIII)( D ), PrP Sc ( E ), and for both ACIII and PrP Sc (Merge)( F ) in CWD infected SGH. Panels A through C , D through F and G through I are the same field of view that are separated into three panels according to the immunofluorescence staining. ToPro®-3 staining of nuclei is indicated by blue fluorescence. Olfactory receptor neurons in the olfactory sensory epithelium (OSE width indicated by white line), and nerve fibers in the subepithelial layer (SE), both express high levels of OMP (A, C and G and I). ACIII is located on the sensory cilia that project from the terminal dendrites of ORNs and its distribution was prominent at the border between the OSE and airway lumen (D and F). The white arrow points to the distal edge of the OSE where it borders the lumen of the nasal airway. Scale bar in A, D and G is 50 µm.

    Techniques Used: Infection, Confocal Microscopy, Marker, Immunofluorescence, Staining, Fluorescence

    2) Product Images from "Timely Synthesis of the Adenovirus Type 5 E1B 55-Kilodalton Protein Is Required for Efficient Genome Replication in Normal Human Cells"

    Article Title: Timely Synthesis of the Adenovirus Type 5 E1B 55-Kilodalton Protein Is Required for Efficient Genome Replication in Normal Human Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.06764-11

    Localization of Mre11, E2 DBP, and the E4 Orf3 proteins in infected HFFs. HFFs at ∼70% confluence were infected with 50 PFU/cell AdEasyE1 (WT), the AdEasyE1Δ2347 mutant (Δ2347), or mock-infected cells (M) for 24 h. They were then processed for immunofluorescence, and Mre11, E2 DBP, and E4 Orf3 were visualized as described in Materials and Methods. The E4 Orf3 protein signal is false-colored in blue. Nuclei stained with DAPI are shown false-colored in cyan. The merged images do not include the nuclear stain.
    Figure Legend Snippet: Localization of Mre11, E2 DBP, and the E4 Orf3 proteins in infected HFFs. HFFs at ∼70% confluence were infected with 50 PFU/cell AdEasyE1 (WT), the AdEasyE1Δ2347 mutant (Δ2347), or mock-infected cells (M) for 24 h. They were then processed for immunofluorescence, and Mre11, E2 DBP, and E4 Orf3 were visualized as described in Materials and Methods. The E4 Orf3 protein signal is false-colored in blue. Nuclei stained with DAPI are shown false-colored in cyan. The merged images do not include the nuclear stain.

    Techniques Used: Infection, Mutagenesis, Immunofluorescence, Staining

    3) Product Images from "Anti-serpin Antibody-mediated Regulation of Proteases in Autoimmune Diabetes *"

    Article Title: Anti-serpin Antibody-mediated Regulation of Proteases in Autoimmune Diabetes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.409664

    Effect of anti-serpin B13 mAb on CD4 and CD19 in pancreas-associated lymphocytes. A , left panels , FACS analysis of CD4 expression in the islets and inguinal lymph nodes of NOD mice treated with control IgG ( n = 4), anti-serpin B13 mAb ( n = 4), the E64
    Figure Legend Snippet: Effect of anti-serpin B13 mAb on CD4 and CD19 in pancreas-associated lymphocytes. A , left panels , FACS analysis of CD4 expression in the islets and inguinal lymph nodes of NOD mice treated with control IgG ( n = 4), anti-serpin B13 mAb ( n = 4), the E64

    Techniques Used: FACS, Expressing, Mouse Assay

    Effect of anti-serpin B13 natural autoantibodies on CD4 in the pancreas-associated lymphocytes. Left panels , analysis of CD4 expression in 4-week-old female NOD mice that had been prescreened for low (SBA low ) or high (SBA high ) secretion of anti-serpin
    Figure Legend Snippet: Effect of anti-serpin B13 natural autoantibodies on CD4 in the pancreas-associated lymphocytes. Left panels , analysis of CD4 expression in 4-week-old female NOD mice that had been prescreened for low (SBA low ) or high (SBA high ) secretion of anti-serpin

    Techniques Used: Expressing, Mouse Assay

    Effect of anti-serpin B13 mAb on protease targets. A , serum-binding activity of serpin B13 in NOD mice isolated from different sources. Left panel , serum samples that were positive ( S1–S3 ; n = 3) or negative ( S4–S6 ; n = 3) for binding
    Figure Legend Snippet: Effect of anti-serpin B13 mAb on protease targets. A , serum-binding activity of serpin B13 in NOD mice isolated from different sources. Left panel , serum samples that were positive ( S1–S3 ; n = 3) or negative ( S4–S6 ; n = 3) for binding

    Techniques Used: Binding Assay, Activity Assay, Mouse Assay, Isolation

    Expression of serpin B13 in the pancreas. Shown is the costaining of frozen pancreatic sections obtained from 6-week-old NOD mouse with anti-serpin B13 mAb and antibodies directed against glucagon ( A ), CD31 ( B ), and keratin-19 ( C ). Staining with the isotype
    Figure Legend Snippet: Expression of serpin B13 in the pancreas. Shown is the costaining of frozen pancreatic sections obtained from 6-week-old NOD mouse with anti-serpin B13 mAb and antibodies directed against glucagon ( A ), CD31 ( B ), and keratin-19 ( C ). Staining with the isotype

    Techniques Used: Expressing, Staining

    4) Product Images from "Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA-Induced Leukemogenesis"

    Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA-Induced Leukemogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00585-16

    Myeloid immortalization of KSL and MP cells by Eya2 . (A) Experimental strategy for myeloid immortalization assays with retroviral transduction using pMYs-IN. (B) Structure of Eya2 and missense mutants. Arrows show primers used in RT-qPCR. (C) Expression of Eya2 and its mutants by Western blotting analyses using anti-Eya2 and anti-α-tubulin (αTub [an internal control]) antibodies (αEya2 and αTub). (D) Expression levels of Eya2 and its mutants by RT-qPCR of colony-forming cells at the end of the first plating in panel A. (E) Myeloid immortalization assays of KSL and MP cells after retroviral transduction. (F to H) Typical morphology of colonies of Eya2 -transduced KSL cells at the third round of plating (F) and typical morphology (G) and immunophenotype (H) of the cells constituting the colonies. Cells were stained with Wright-Giemsa stain. (I) Localization of Eya2 in the FLAG-tagged Eya2 -immortalized KSL cells analyzed by immunofluorescent confocal microscopy. Alexa Fluor 568-conjugated secondary antibody reacting with anti-DDDDK-tag antibody (αDDDDK) in the immortalized cells visualized their cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and a merged image is displayed (bottom). Magnifications (bar lengths): F, ×40 (200 μm); G, ×400 (20 μm); I, ×400 (30 μm). Bar graphs show means ± SD from three independent experiments.
    Figure Legend Snippet: Myeloid immortalization of KSL and MP cells by Eya2 . (A) Experimental strategy for myeloid immortalization assays with retroviral transduction using pMYs-IN. (B) Structure of Eya2 and missense mutants. Arrows show primers used in RT-qPCR. (C) Expression of Eya2 and its mutants by Western blotting analyses using anti-Eya2 and anti-α-tubulin (αTub [an internal control]) antibodies (αEya2 and αTub). (D) Expression levels of Eya2 and its mutants by RT-qPCR of colony-forming cells at the end of the first plating in panel A. (E) Myeloid immortalization assays of KSL and MP cells after retroviral transduction. (F to H) Typical morphology of colonies of Eya2 -transduced KSL cells at the third round of plating (F) and typical morphology (G) and immunophenotype (H) of the cells constituting the colonies. Cells were stained with Wright-Giemsa stain. (I) Localization of Eya2 in the FLAG-tagged Eya2 -immortalized KSL cells analyzed by immunofluorescent confocal microscopy. Alexa Fluor 568-conjugated secondary antibody reacting with anti-DDDDK-tag antibody (αDDDDK) in the immortalized cells visualized their cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and a merged image is displayed (bottom). Magnifications (bar lengths): F, ×40 (200 μm); G, ×400 (20 μm); I, ×400 (30 μm). Bar graphs show means ± SD from three independent experiments.

    Techniques Used: Transduction, Quantitative RT-PCR, Expressing, Western Blot, Staining, Giemsa Stain, Confocal Microscopy

    Inducible immortalization of KSL and MP cells by ER-Eya2 . (A) Experimental strategy for myeloid immortalization assays of KSL and MP cells with retroviral transduction of the ER-Eya2 fusion gene using pMYs-IN. ER , estrogen receptor gene; 4OHT, 4-hydroxytamoxifen. (B) Structure of Eya2 and ER-Eya2. LBD, mutant ligand-binding domain of the mouse ER. (C) Expression of ER-Eya2 by Western blotting using anti-Eya2 (αEya2 [top]), anti-ER antibody (αER [middle]), and anti-α-tubulin (αTub [an internal control; bottom]). (D) Myeloid immortalization assays of ER-Eya2 -transduced cells in the presence or absence of 4OHT. (E) 4OHT-dependent clonogenicity of cells inducibly immortalized by ER-Eya2 . (F) Localization of ER-Eya2 in the inducibly immortalized KSL cells analyzed by immunofluorescent confocal microscopy, following a 3-day culture in the presence (left panels) and absence (right panels) of 4OHT. Alexa Fluor 568-conjugated secondary antibody reacting with anti-ER in the immortalized cells visualized its cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and merged images are displayed (bottom). Magnification (bar length), ×400 (30 μm). Bar graphs show the means ± SD from three independent experiments.
    Figure Legend Snippet: Inducible immortalization of KSL and MP cells by ER-Eya2 . (A) Experimental strategy for myeloid immortalization assays of KSL and MP cells with retroviral transduction of the ER-Eya2 fusion gene using pMYs-IN. ER , estrogen receptor gene; 4OHT, 4-hydroxytamoxifen. (B) Structure of Eya2 and ER-Eya2. LBD, mutant ligand-binding domain of the mouse ER. (C) Expression of ER-Eya2 by Western blotting using anti-Eya2 (αEya2 [top]), anti-ER antibody (αER [middle]), and anti-α-tubulin (αTub [an internal control; bottom]). (D) Myeloid immortalization assays of ER-Eya2 -transduced cells in the presence or absence of 4OHT. (E) 4OHT-dependent clonogenicity of cells inducibly immortalized by ER-Eya2 . (F) Localization of ER-Eya2 in the inducibly immortalized KSL cells analyzed by immunofluorescent confocal microscopy, following a 3-day culture in the presence (left panels) and absence (right panels) of 4OHT. Alexa Fluor 568-conjugated secondary antibody reacting with anti-ER in the immortalized cells visualized its cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and merged images are displayed (bottom). Magnification (bar length), ×400 (30 μm). Bar graphs show the means ± SD from three independent experiments.

    Techniques Used: Transduction, Mutagenesis, Ligand Binding Assay, Expressing, Western Blot, Confocal Microscopy

    5) Product Images from "GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production"

    Article Title: GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-05-0270

    BACE1 phospho-mutants show differences in steady-state distribution and cell-surface expression in HeLa cells. (A) Schematic representation of BACE1 showing the luminal, transmembrane, and cytoplasmic domains. Ser498 in BACE1 was substituted with either an alanine or aspartate to mimic an unphosphorylated (green) or phosphorylated (blue) form of BACE1. (B) Immunoblotting of cell extracts of HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants for 24 h and probed with rabbit anti-pSer498 BACE1 antibodies, rabbit anti-BACE1 antibodies, and mouse anti–α-tubulin antibodies, using a chemiluminescence detection system. (C, D) Confocal microscopic images of fixed and permeabilized HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants and stained with rabbit polyclonal anti-human BACE1 antibodies (red) and mouse monoclonal antibodies to (C) Rab 11 or (D) EEA1 (green). Higher magnifications of the merge images are also shown. Bars represent 10 µm. (E–H) Percentage of BACE1 at the early endosomes, recycling endosomes, late endosomes, or the TGN was calculated from the percentage of total BACE1 pixels that overlapped with (E) Rab11, (F) EEA1, (G) CD63, or (H) golgin97, respectively. All calculations were performed using the OBCOL plug-in on ImageJ ( n = 15 for each marker from three independent experiments). (I) PulSA analyses. HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants were harvested, fixed, and permeabilized; stained with rabbit polyclonal anti-human BACE1 antibodies; and analzyed by flow cytometry (FACS) for the pulse width of the fluorescent signal. Histograms show the mean pulse width and SEM from three independent experiments. (J) Cell-surface expression of HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants. Viable cells in suspension were incubated with anti-BACE1 antibodies on ice for 30 min, fixed in 4% PFA, stained with Alexa488-conjugated IgG, and analyzed by FACS. Histograms shows the mean fluorescence intensity of cell-surface BACE1 normalized for the total BACE1 protein level for each BACE1 variant. Shown is the mean and SEM for three independent experiments. Bars represent 10 µm. (E–J) * p
    Figure Legend Snippet: BACE1 phospho-mutants show differences in steady-state distribution and cell-surface expression in HeLa cells. (A) Schematic representation of BACE1 showing the luminal, transmembrane, and cytoplasmic domains. Ser498 in BACE1 was substituted with either an alanine or aspartate to mimic an unphosphorylated (green) or phosphorylated (blue) form of BACE1. (B) Immunoblotting of cell extracts of HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants for 24 h and probed with rabbit anti-pSer498 BACE1 antibodies, rabbit anti-BACE1 antibodies, and mouse anti–α-tubulin antibodies, using a chemiluminescence detection system. (C, D) Confocal microscopic images of fixed and permeabilized HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants and stained with rabbit polyclonal anti-human BACE1 antibodies (red) and mouse monoclonal antibodies to (C) Rab 11 or (D) EEA1 (green). Higher magnifications of the merge images are also shown. Bars represent 10 µm. (E–H) Percentage of BACE1 at the early endosomes, recycling endosomes, late endosomes, or the TGN was calculated from the percentage of total BACE1 pixels that overlapped with (E) Rab11, (F) EEA1, (G) CD63, or (H) golgin97, respectively. All calculations were performed using the OBCOL plug-in on ImageJ ( n = 15 for each marker from three independent experiments). (I) PulSA analyses. HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants were harvested, fixed, and permeabilized; stained with rabbit polyclonal anti-human BACE1 antibodies; and analzyed by flow cytometry (FACS) for the pulse width of the fluorescent signal. Histograms show the mean pulse width and SEM from three independent experiments. (J) Cell-surface expression of HeLa cells transfected with either wtBACE1 or BACE1 phosphomutants. Viable cells in suspension were incubated with anti-BACE1 antibodies on ice for 30 min, fixed in 4% PFA, stained with Alexa488-conjugated IgG, and analyzed by FACS. Histograms shows the mean fluorescence intensity of cell-surface BACE1 normalized for the total BACE1 protein level for each BACE1 variant. Shown is the mean and SEM for three independent experiments. Bars represent 10 µm. (E–J) * p

    Techniques Used: Expressing, Transfection, Staining, Marker, Flow Cytometry, Cytometry, FACS, Incubation, Fluorescence, Variant Assay

    6) Product Images from "Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183"

    Article Title: Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190407

    Characterization of the novel ubiquitin ligase RNF183. ]. Blue box, transmembrane domain; red triangle, RING-finger domain; purple box, low complexity sequence. The number at right indicates the peptide length. (C) In vitro auto-ubiquitination of wild type and mutant RNF183. In vitro transcribed/translated V5-tagged RNF183 tagged was mixed and incubated with recombinant E1, E2, and HA-ubiquitin. The reaction mixture was immunoprecipitated with an anti-V5 antibody and subjected to Western blotting with anti-polyubiquitin ( upper panel) and anti-V5 antibodies ( lower panel). WT, wild type; ΔR, RING-finger domain deletion mutant; CS, Cys13-, and Cys16-to-Ser point mutations in the RING domain; IP, immunoprecipitation; Ub, ubiquitin. Asterisk indicates the immunoglobulin heavy chain. (D) In vitro auto-ubiquitination of RNF183 in the absence of each component. (E) Subcellular localization of RNF183. HeLa cells stably transfected with RNF183-V5 ( red ) were subjected to immunofluorescence staining with various antibodies for organelle makers (Calnexin, GM130, EEA1, Rab7 and LAMP1; green ) and DAPI for nuclear staining ( blue ).
    Figure Legend Snippet: Characterization of the novel ubiquitin ligase RNF183. ]. Blue box, transmembrane domain; red triangle, RING-finger domain; purple box, low complexity sequence. The number at right indicates the peptide length. (C) In vitro auto-ubiquitination of wild type and mutant RNF183. In vitro transcribed/translated V5-tagged RNF183 tagged was mixed and incubated with recombinant E1, E2, and HA-ubiquitin. The reaction mixture was immunoprecipitated with an anti-V5 antibody and subjected to Western blotting with anti-polyubiquitin ( upper panel) and anti-V5 antibodies ( lower panel). WT, wild type; ΔR, RING-finger domain deletion mutant; CS, Cys13-, and Cys16-to-Ser point mutations in the RING domain; IP, immunoprecipitation; Ub, ubiquitin. Asterisk indicates the immunoglobulin heavy chain. (D) In vitro auto-ubiquitination of RNF183 in the absence of each component. (E) Subcellular localization of RNF183. HeLa cells stably transfected with RNF183-V5 ( red ) were subjected to immunofluorescence staining with various antibodies for organelle makers (Calnexin, GM130, EEA1, Rab7 and LAMP1; green ) and DAPI for nuclear staining ( blue ).

    Techniques Used: Sequencing, In Vitro, Mutagenesis, Incubation, Recombinant, Immunoprecipitation, Western Blot, Stable Transfection, Transfection, Immunofluorescence, Staining

    Effects of Sec16 on RNF183 subcellular localization. (A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 ( green ) and anti-calnexin, GM130, or LAMP1 ( red ) antibodies, and DAPI ( blue ). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC ( top panels) or Sec16A ( middle and bottom panels) siRNAs. At 36 h after transfection, cells were incubated with ( bottom panels) or without ( top and middle panels) 10 μM MG132 for 12 h.
    Figure Legend Snippet: Effects of Sec16 on RNF183 subcellular localization. (A) Effect of Sec16A downregulation on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, 5th panels) or Sec16A (2nd, 4th, 6th panels) siRNAs. At 48 h after transfection, cells were subjected to immunofluorescence staining with anti-V5 ( green ) and anti-calnexin, GM130, or LAMP1 ( red ) antibodies, and DAPI ( blue ). (B) Effect of proteasome inhibition on RNF183 subcellular localization. HeLa cells stably expressing RNF183-V5 were transfected with NC ( top panels) or Sec16A ( middle and bottom panels) siRNAs. At 36 h after transfection, cells were incubated with ( bottom panels) or without ( top and middle panels) 10 μM MG132 for 12 h.

    Techniques Used: Stable Transfection, Expressing, Transfection, Immunofluorescence, Staining, Inhibition, Incubation

    Effects of Sec16 on other ubiquitin ligases. (A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody ( top panel) and anti-V5 ( second panel) or anti-myc antibodies ( third panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p
    Figure Legend Snippet: Effects of Sec16 on other ubiquitin ligases. (A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody ( top panel) and anti-V5 ( second panel) or anti-myc antibodies ( third panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p

    Techniques Used: Stable Transfection, Immunoprecipitation, Negative Control, Western Blot, Expressing, Transfection

    Effects of Sec16 on RNF183 protein stability. (A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p
    Figure Legend Snippet: Effects of Sec16 on RNF183 protein stability. (A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p

    Techniques Used: Stable Transfection, Expressing, Transfection, Western Blot

    7) Product Images from "Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells"

    Article Title: Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells

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

    doi: 10.4049/jimmunol.1002835

    Knockdown of EC JAM-1 inhibits chemokine-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or two different siRNAs targeting JAM-1 (JAM-1 siRNA-5 and JAM-1 siRNA-6), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen, and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–JAM-1 (thick lines) demonstrating effective knockdown. B , Left lower panel (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Right lower panel on (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of three (VB2 − ) and two (VB2 + ) separate experiments using T cells from different donors. * p
    Figure Legend Snippet: Knockdown of EC JAM-1 inhibits chemokine-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or two different siRNAs targeting JAM-1 (JAM-1 siRNA-5 and JAM-1 siRNA-6), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen, and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–JAM-1 (thick lines) demonstrating effective knockdown. B , Left lower panel (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Right lower panel on (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of three (VB2 − ) and two (VB2 + ) separate experiments using T cells from different donors. * p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining

    Blocking of T cell LFA-1 but not Mac-1 inhibits TCR-dependent TEM of EM CD4 + T cells. A , Mac-1 is expressed on EM CD4 + T cells. Contour plots of FACS analysis of CD45RA − CD4 + T cells stained with isotype-matched control PE- and FITC-conjugated IgG ( left plot ) or PE-conjugated anti-CD11b and FITC-conjugated anti-CCR7 ( right plot ). Note that there is a significant proportion of CD11b + cells in the CCR7 low (i.e., EM) population. B , TEM assays. EM CD4 + T cells were preincubated with isotype control IgG (control), anti–LFA-1 (LFA-1), or anti–Mac-1 (Mac-1) blocking mAb 30 min prior to flow TEM on TNF-treated CIITA-transduced HDMECs overlaid with TSST-1. Panel on the left (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Panel on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data pooled from three separate experiments using T cells isolated from three different donors. *** p
    Figure Legend Snippet: Blocking of T cell LFA-1 but not Mac-1 inhibits TCR-dependent TEM of EM CD4 + T cells. A , Mac-1 is expressed on EM CD4 + T cells. Contour plots of FACS analysis of CD45RA − CD4 + T cells stained with isotype-matched control PE- and FITC-conjugated IgG ( left plot ) or PE-conjugated anti-CD11b and FITC-conjugated anti-CCR7 ( right plot ). Note that there is a significant proportion of CD11b + cells in the CCR7 low (i.e., EM) population. B , TEM assays. EM CD4 + T cells were preincubated with isotype control IgG (control), anti–LFA-1 (LFA-1), or anti–Mac-1 (Mac-1) blocking mAb 30 min prior to flow TEM on TNF-treated CIITA-transduced HDMECs overlaid with TSST-1. Panel on the left (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Panel on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data pooled from three separate experiments using T cells isolated from three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, FACS, Staining, Flow Cytometry, Isolation

    Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p
    Figure Legend Snippet: Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, Flow Cytometry

    Blocking of T cell DNAM-1 and/or Tactile inhibits TCR-dependent TEM of EM CD4 + T cells. A , Contour plots of FACS analysis of total CD4 + T cells stained with FITC-conjugated IgG (IgG–FITC) and PE-conjugated IgG (IgG–PE, upper left ) or FITC-conjugated anti-CCR7 (CCR7–FITC) and PE-conjugated anti–DNAM-1 (DNAM-1–PE, lower left ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed IgG (IgG-647, upper right ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed anti-Tactile (Tactile-647, lower right ). Note the distinct population of cells that are DNAM-1 high, CCR7 low or Tactile high, CCR7 low in the middle , left , and right plots , respectively. Lower histograms show overlays of the isotype-matched control IgG and anti–DNAM-1 ( left ) or anti-Tactile ( right ) plots. B , EM CD4 + T cells were preincubated with isotype-matched control IgG (control), anti–DNAM-1 (DNAM-1), anti-Tactile (Tactile), and both anti–DNAM-1 and anti-Tactile blocking mAbs (DNAM-1+Tactile) prior to flow TEM. Left panel shows TEM of Vβ2TCR − cells at 15 min. Right panel shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) and four (VB2 + ) separate experiments using T cells isolated from different donors. ** p
    Figure Legend Snippet: Blocking of T cell DNAM-1 and/or Tactile inhibits TCR-dependent TEM of EM CD4 + T cells. A , Contour plots of FACS analysis of total CD4 + T cells stained with FITC-conjugated IgG (IgG–FITC) and PE-conjugated IgG (IgG–PE, upper left ) or FITC-conjugated anti-CCR7 (CCR7–FITC) and PE-conjugated anti–DNAM-1 (DNAM-1–PE, lower left ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed IgG (IgG-647, upper right ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed anti-Tactile (Tactile-647, lower right ). Note the distinct population of cells that are DNAM-1 high, CCR7 low or Tactile high, CCR7 low in the middle , left , and right plots , respectively. Lower histograms show overlays of the isotype-matched control IgG and anti–DNAM-1 ( left ) or anti-Tactile ( right ) plots. B , EM CD4 + T cells were preincubated with isotype-matched control IgG (control), anti–DNAM-1 (DNAM-1), anti-Tactile (Tactile), and both anti–DNAM-1 and anti-Tactile blocking mAbs (DNAM-1+Tactile) prior to flow TEM. Left panel shows TEM of Vβ2TCR − cells at 15 min. Right panel shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) and four (VB2 + ) separate experiments using T cells isolated from different donors. ** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, FACS, Staining, Flow Cytometry, Isolation

    Knockdown of EC CD99 inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMEC were transfected with control siRNA or two different siRNAs targeting CD99 (CD99 siRNA-2 and CD99 siRNA-5), treated with TNF, analyzed by FACS ( A ) or overlaid with TSST-1 superantigen (recognized by those T cells with the germline-encoded Vβ2 segment in the TCR), and used in flow TEM assays with EM CD4 + T cells ( B ). A , FACS plots of HDMECs stained with isotype-matched control IgG (thin lines) or anti-CD99 or -CD31 (thick lines in left panels and right panels , respectively) demonstrating knockdown of CD99 without reduction of CD31 expression. B , TEM assays. Graph on the left (VB2 − ) shows TEM of Vβ2TCR − cells (i.e., those T cells with TCR that are not activated by TSST-1) at 15 min. Graph on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) or three (VB2 + ) separate experiments using T cells from different donors. *** p
    Figure Legend Snippet: Knockdown of EC CD99 inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMEC were transfected with control siRNA or two different siRNAs targeting CD99 (CD99 siRNA-2 and CD99 siRNA-5), treated with TNF, analyzed by FACS ( A ) or overlaid with TSST-1 superantigen (recognized by those T cells with the germline-encoded Vβ2 segment in the TCR), and used in flow TEM assays with EM CD4 + T cells ( B ). A , FACS plots of HDMECs stained with isotype-matched control IgG (thin lines) or anti-CD99 or -CD31 (thick lines in left panels and right panels , respectively) demonstrating knockdown of CD99 without reduction of CD31 expression. B , TEM assays. Graph on the left (VB2 − ) shows TEM of Vβ2TCR − cells (i.e., those T cells with TCR that are not activated by TSST-1) at 15 min. Graph on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) or three (VB2 + ) separate experiments using T cells from different donors. *** p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining, Expressing

    8) Product Images from "NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes"

    Article Title: NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.03293-14

    Chemically diverse NS5A inhibitors block NS5A hyperphosphorylation. 10A-IFN cells were transfected with the construct pCD-BlaRep-wt (NS3-5B wt) (A) or pCD-BlaRep-K@67 (NS3-5B 5A-K@67) or pCD-BlaRep-K@67-Y93H (NS3-5B 5A-K@67, Y93H) (B) and Con1 HCV polyprotein
    Figure Legend Snippet: Chemically diverse NS5A inhibitors block NS5A hyperphosphorylation. 10A-IFN cells were transfected with the construct pCD-BlaRep-wt (NS3-5B wt) (A) or pCD-BlaRep-K@67 (NS3-5B 5A-K@67) or pCD-BlaRep-K@67-Y93H (NS3-5B 5A-K@67, Y93H) (B) and Con1 HCV polyprotein

    Techniques Used: Blocking Assay, Transfection, Construct

    NS5A inhibitors promote formation of the large-cluster NS5A phenotype. (A) IF staining of NS5A of Huh7.5-10A and Huh7.5-10A-Y93H cells treated for 8 h with 0.2% DMSO as a control or with the indicated compounds at 20× the EC 50 : DCV, 250 pM; I-7,
    Figure Legend Snippet: NS5A inhibitors promote formation of the large-cluster NS5A phenotype. (A) IF staining of NS5A of Huh7.5-10A and Huh7.5-10A-Y93H cells treated for 8 h with 0.2% DMSO as a control or with the indicated compounds at 20× the EC 50 : DCV, 250 pM; I-7,

    Techniques Used: Staining

    NS5A inhibitors impair NS5A-PI4KIIIα protein complex formation. (A) 10A-IFN cells were cotransfected with the constructs pEF1A-PIK4CA (PI4KIIIα) and pCD-BlaRep-K@67 (NS3-5B 5A-K@67) or pCD-BlaRep-K@67-Y93H (NS3-5B 5A-K@67, Y93H), and the
    Figure Legend Snippet: NS5A inhibitors impair NS5A-PI4KIIIα protein complex formation. (A) 10A-IFN cells were cotransfected with the constructs pEF1A-PIK4CA (PI4KIIIα) and pCD-BlaRep-K@67 (NS3-5B 5A-K@67) or pCD-BlaRep-K@67-Y93H (NS3-5B 5A-K@67, Y93H), and the

    Techniques Used: Construct

    NS5A inhibitors interfere with the enrichment of PI4P in HCV replication membranes. (A) IF staining of PI4P and NS5A in Huh7.5-10A and Huh7.5-10A-Y93H cells treated for 8 h with 0.2% DMSO as a control or with the indicated compounds at 20× the
    Figure Legend Snippet: NS5A inhibitors interfere with the enrichment of PI4P in HCV replication membranes. (A) IF staining of PI4P and NS5A in Huh7.5-10A and Huh7.5-10A-Y93H cells treated for 8 h with 0.2% DMSO as a control or with the indicated compounds at 20× the

    Techniques Used: Staining

    Chemically diverse NS5A inhibitors block NS5A hyperphosphorylation.
    Figure Legend Snippet: Chemically diverse NS5A inhibitors block NS5A hyperphosphorylation.

    Techniques Used: Blocking Assay

    Both PI4KIIIα and NS5A inhibitors reduce cholesterol concentration in the HCV-induced membranous web. Fluorescence staining of intracellular PI4P and unesterified cholesterol. The detection of PI4P (green) and Filipin (red) was performed as detailed
    Figure Legend Snippet: Both PI4KIIIα and NS5A inhibitors reduce cholesterol concentration in the HCV-induced membranous web. Fluorescence staining of intracellular PI4P and unesterified cholesterol. The detection of PI4P (green) and Filipin (red) was performed as detailed

    Techniques Used: Concentration Assay, Fluorescence, Staining

    9) Product Images from "Hepatitis E Virus Genotype 1 Infection of Swine Kidney Cells In Vitro Is Inhibited at Multiple Levels"

    Article Title: Hepatitis E Virus Genotype 1 Infection of Swine Kidney Cells In Vitro Is Inhibited at Multiple Levels

    Journal: Journal of Virology

    doi: 10.1128/JVI.02205-13

    Flow cytometry of transfected cells stained separately for the ORF2 or ORF3 protein. HepG2/C3A and LLC-PK cells electroporated with transcripts from P6 (A), Sar55 (B), or Sar/S17 (C, D) were plated in 6 wells of a 6-well culture plate, incubated at 34.5°C, and harvested 7 (A, B) or 5 (C, D) days later. Cells in triplicate wells were stained for the ORF2 protein, followed by goat anti-human IgG labeled with Alexa Fluor 488 (open bars); cells in the other 3 wells were stained for the ORF3 protein, followed by goat anti-rabbit IgG also labeled with Alexa Fluor 488 (hatched bars). Flow cytometry was performed with the same settings for all samples. (A to C) Mean percentage of positive cells, in triplicate; (D) mean of the geometric mean fluorescence intensity in the same triplicate samples assayed for panel C. Shaded bars, ORF2; dotted bars, ORF3. Error bars indicate standard deviations. P values were determined by Student's t test; P values of less than 0.05 were statistically significant.
    Figure Legend Snippet: Flow cytometry of transfected cells stained separately for the ORF2 or ORF3 protein. HepG2/C3A and LLC-PK cells electroporated with transcripts from P6 (A), Sar55 (B), or Sar/S17 (C, D) were plated in 6 wells of a 6-well culture plate, incubated at 34.5°C, and harvested 7 (A, B) or 5 (C, D) days later. Cells in triplicate wells were stained for the ORF2 protein, followed by goat anti-human IgG labeled with Alexa Fluor 488 (open bars); cells in the other 3 wells were stained for the ORF3 protein, followed by goat anti-rabbit IgG also labeled with Alexa Fluor 488 (hatched bars). Flow cytometry was performed with the same settings for all samples. (A to C) Mean percentage of positive cells, in triplicate; (D) mean of the geometric mean fluorescence intensity in the same triplicate samples assayed for panel C. Shaded bars, ORF2; dotted bars, ORF3. Error bars indicate standard deviations. P values were determined by Student's t test; P values of less than 0.05 were statistically significant.

    Techniques Used: Flow Cytometry, Cytometry, Transfection, Staining, Incubation, Labeling, Fluorescence

    10) Product Images from "Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration"

    Article Title: Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201505298

    Activation status of microglia infiltrating the outer nuclear layer (ONL) and the exposure of phosphatidylserine (PS) on ONL photoreceptors Microglia infiltrating the outer nuclear layer (ONL) of the rd10 retina during rod photoreceptor degeneration demonstrate markers of activation. At P18, Iba1 + microglia (green, arrow) in the outer plexiform layer were negative for TSPO (red), an activation marker. At P22, Iba1 + microglia (arrow) infiltrated the ONL and acquired TSPO immunopositivity, indicating their activated status. Scale bars, 25 μm. Phosphatidylserine (PS) exposure in ONL nuclei of unfixed cryosections of rd10 retina during rod degeneration. PS exposure in the ONL was monitored in unfixed frozen sections using fluorescently conjugated annexin V which binds cell-surface PS. While minimal annexin V staining was evident in the ONL in P18 wild-type (top row) and P18 rd10 (middle row) retinas in which rod degeneration is absent, staining was prominent in P21 rd10 retina during rod degeneration (bottom row). Scale bar, 40 μm.
    Figure Legend Snippet: Activation status of microglia infiltrating the outer nuclear layer (ONL) and the exposure of phosphatidylserine (PS) on ONL photoreceptors Microglia infiltrating the outer nuclear layer (ONL) of the rd10 retina during rod photoreceptor degeneration demonstrate markers of activation. At P18, Iba1 + microglia (green, arrow) in the outer plexiform layer were negative for TSPO (red), an activation marker. At P22, Iba1 + microglia (arrow) infiltrated the ONL and acquired TSPO immunopositivity, indicating their activated status. Scale bars, 25 μm. Phosphatidylserine (PS) exposure in ONL nuclei of unfixed cryosections of rd10 retina during rod degeneration. PS exposure in the ONL was monitored in unfixed frozen sections using fluorescently conjugated annexin V which binds cell-surface PS. While minimal annexin V staining was evident in the ONL in P18 wild-type (top row) and P18 rd10 (middle row) retinas in which rod degeneration is absent, staining was prominent in P21 rd10 retina during rod degeneration (bottom row). Scale bar, 40 μm.

    Techniques Used: Activation Assay, Marker, Staining

    11) Product Images from "Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity"

    Article Title: Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04334-1

    Validation and spatial integration of two distinct luminal cell types. a Immunofluorescence analysis of NY-BR-1 protein expression (green) in combination with basal marker SLPI (red) and DNA stain using DAPI (blue) within tissue sections from primary human reduction mammoplasty samples revealed that NY-BR-1 and SLPI are markers for distinct luminal subpopulations. b – e Immunofluorescence analysis of NY-BR-1 and SLPI (red) protein expression with: hormone receptors for estrogen receptor ( b ), progesterone ( c ), and androgen ( d ) and proliferation marker Ki67 e in green. f Summary of hormone receptor and proliferation marker expression in L1 and L2 cells. g Violin plot showing expression of KRT8 in the luminal subpopulations, higher expression is seen in the luminal L1.1 and L1.2 subpopulation. h Sample frame for detection of KRT8 protein content from individual cells using single cell Western blot following detection using microarray scanner. i for violin plots displaying expression of relevant hormone receptors as well as proliferation and luminal progenitor markers. All scale bars = 25 µm
    Figure Legend Snippet: Validation and spatial integration of two distinct luminal cell types. a Immunofluorescence analysis of NY-BR-1 protein expression (green) in combination with basal marker SLPI (red) and DNA stain using DAPI (blue) within tissue sections from primary human reduction mammoplasty samples revealed that NY-BR-1 and SLPI are markers for distinct luminal subpopulations. b – e Immunofluorescence analysis of NY-BR-1 and SLPI (red) protein expression with: hormone receptors for estrogen receptor ( b ), progesterone ( c ), and androgen ( d ) and proliferation marker Ki67 e in green. f Summary of hormone receptor and proliferation marker expression in L1 and L2 cells. g Violin plot showing expression of KRT8 in the luminal subpopulations, higher expression is seen in the luminal L1.1 and L1.2 subpopulation. h Sample frame for detection of KRT8 protein content from individual cells using single cell Western blot following detection using microarray scanner. i for violin plots displaying expression of relevant hormone receptors as well as proliferation and luminal progenitor markers. All scale bars = 25 µm

    Techniques Used: Immunofluorescence, Expressing, Marker, Staining, Western Blot, Microarray

    12) Product Images from "Intra-articular injection of synthetic microRNA-210 accelerates avascular meniscal healing in rat medial meniscal injured model"

    Article Title: Intra-articular injection of synthetic microRNA-210 accelerates avascular meniscal healing in rat medial meniscal injured model

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-014-0488-y

    Immunohistochemistry of meniscus at 4 weeks after intra-articular injection. (A) Immunohistochemistry of vascular endothelial growth factor (VEGF; upper) and fibroblast growth factor-2 (FGF2; lower). VEGF and FGF2 expressed intensely on the surface of the meniscus, around the injured site and in the red zone in the miR-210 group compared with the control group. Arrow, injured site. Bar = 100 μm. (B) Immunohistochemistry of type 2 collagen. Type 2 collagen expression was observed around the injured site of the meniscus in the miR-210 group compared with the control group. Arrow, injured site. Bar = 100 μm. (C) Isolectin B4 staining and the number of blood vessels. Newly formed vessels were observed around the injured site in the miR-210 group, while little blood vessels were observed in the control. White arrow, injured site; yellow arrow, blood vessels. Bar = 100 μm. Number of blood vessels in the miR-210 group was significantly higher than that in the control group (* P
    Figure Legend Snippet: Immunohistochemistry of meniscus at 4 weeks after intra-articular injection. (A) Immunohistochemistry of vascular endothelial growth factor (VEGF; upper) and fibroblast growth factor-2 (FGF2; lower). VEGF and FGF2 expressed intensely on the surface of the meniscus, around the injured site and in the red zone in the miR-210 group compared with the control group. Arrow, injured site. Bar = 100 μm. (B) Immunohistochemistry of type 2 collagen. Type 2 collagen expression was observed around the injured site of the meniscus in the miR-210 group compared with the control group. Arrow, injured site. Bar = 100 μm. (C) Isolectin B4 staining and the number of blood vessels. Newly formed vessels were observed around the injured site in the miR-210 group, while little blood vessels were observed in the control. White arrow, injured site; yellow arrow, blood vessels. Bar = 100 μm. Number of blood vessels in the miR-210 group was significantly higher than that in the control group (* P

    Techniques Used: Immunohistochemistry, Injection, Expressing, Staining

    Gene expression analysis by real-time PCR in the meniscus at 4 weeks following intraarticular injection. Expression of mature miR-210, collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) was examined using real-time PCR. Expression of miR-210, Col2a1, VEGF and FGF2 in the miR-210 group was significantly higher than that in the control and normal groups (* P
    Figure Legend Snippet: Gene expression analysis by real-time PCR in the meniscus at 4 weeks following intraarticular injection. Expression of mature miR-210, collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) was examined using real-time PCR. Expression of miR-210, Col2a1, VEGF and FGF2 in the miR-210 group was significantly higher than that in the control and normal groups (* P

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

    Gene expression analyses in inner meniscus cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of inner meniscus cells. Expression of only Col2a1 was significantly higher than that in the control group (* P
    Figure Legend Snippet: Gene expression analyses in inner meniscus cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of inner meniscus cells. Expression of only Col2a1 was significantly higher than that in the control group (* P

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, In Vitro, Transfection

    Gene expression analyses in synovial cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of synovial cells. Expression of Col2a1 was not detected in both groups. Expression of VEGF and FGF2 was significantly higher than in the control group (* P
    Figure Legend Snippet: Gene expression analyses in synovial cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of synovial cells. Expression of Col2a1 was not detected in both groups. Expression of VEGF and FGF2 was significantly higher than in the control group (* P

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, In Vitro, Transfection

    13) Product Images from "Modulation of Glucose Takeup by Glucose Transport on the Isolated OHCs"

    Article Title: Modulation of Glucose Takeup by Glucose Transport on the Isolated OHCs

    Journal: Neural Plasticity

    doi: 10.1155/2018/7513217

    Basal staining for GLUT-4 may result from intracellular penetration of the antibody. OHCs were costained using anti-GLUT-4 antibodies and di-8-ANEPPS: (a) GLUT-4 staining; (b) di-8-ANEPPS staining; (c) merged image; (d) high magnification; (e) Nomarski image. Scale bar, 10 μ m in all images except the inset (2 μ m).
    Figure Legend Snippet: Basal staining for GLUT-4 may result from intracellular penetration of the antibody. OHCs were costained using anti-GLUT-4 antibodies and di-8-ANEPPS: (a) GLUT-4 staining; (b) di-8-ANEPPS staining; (c) merged image; (d) high magnification; (e) Nomarski image. Scale bar, 10 μ m in all images except the inset (2 μ m).

    Techniques Used: Staining

    14) Product Images from "Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells"

    Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells

    Journal: Oncogene

    doi: 10.1038/onc.2014.97

    Coordinate regulation of EMT by UBQLN1 and ZEB1 (a) Loss of Zeb1 increases expression of UBQLN1 and increases epithelial markers in A549 and H358 cells. Western blot analysis of ZEB1, UBQLN1 and EMT markers in A549 and H358 cells. Cells were transfected with either non-targeting siRNA (siNT) or with siRNA targeting ZEB1 (siZEB1). After 72 hrs of transfection, cells were harvested and subjected to western blot for protein expression analysis for UBQLN1, ZEB1 along with other EMT markers (b) UBQLN1 loss requires ZEB1 to induce EMT in A549 and H358 cells. Cells were transfected with non-targeting siRNA, with siUBQLN1, siZEB1 or the combination of siUBQLN1 and siZEB1. After 72 hrs of transfection, cells were harvested. Western blot analysis confirming knockdown of UBQLN1 and ZEB1 along with different EMT markers. (c) Fluorescence staining for E-cadherin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1), siZeb1 or combination of siU1 and siZeb1, cells were trypsinized and plated on chamber slides and stained for E-cadherin. i, iii, v and vii: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv, vi and viii: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (d) A549 cells were prepared as described in (c) and F-actin was detected with Alexa Fluor 568 Phalloidin (red) with 60x objective. Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.
    Figure Legend Snippet: Coordinate regulation of EMT by UBQLN1 and ZEB1 (a) Loss of Zeb1 increases expression of UBQLN1 and increases epithelial markers in A549 and H358 cells. Western blot analysis of ZEB1, UBQLN1 and EMT markers in A549 and H358 cells. Cells were transfected with either non-targeting siRNA (siNT) or with siRNA targeting ZEB1 (siZEB1). After 72 hrs of transfection, cells were harvested and subjected to western blot for protein expression analysis for UBQLN1, ZEB1 along with other EMT markers (b) UBQLN1 loss requires ZEB1 to induce EMT in A549 and H358 cells. Cells were transfected with non-targeting siRNA, with siUBQLN1, siZEB1 or the combination of siUBQLN1 and siZEB1. After 72 hrs of transfection, cells were harvested. Western blot analysis confirming knockdown of UBQLN1 and ZEB1 along with different EMT markers. (c) Fluorescence staining for E-cadherin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1), siZeb1 or combination of siU1 and siZeb1, cells were trypsinized and plated on chamber slides and stained for E-cadherin. i, iii, v and vii: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv, vi and viii: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (d) A549 cells were prepared as described in (c) and F-actin was detected with Alexa Fluor 568 Phalloidin (red) with 60x objective. Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.

    Techniques Used: Expressing, Western Blot, Transfection, Fluorescence, Staining

    Loss of UBQLN1 induces EMT (a) UBQLN1 loss induces EMT in A549 and H358 cells. A549 and H358 cells were transfected with either with non-targeting siRNA (siNT) or siRNAs targeting UBQLN1 (siU1, siU1-2). After 72 hrs of transfection cells were harvested and analyzed for protein expression using the indicated antibodies. (b) Fluorescence staining for E-cadherin and Vimentin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1 and siU1-2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective E-Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (c) Cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows. (d) Table indicating the fold change of mRNA following siRNA mediated knockdown of UBQLN1 for EMT-associated genes, as compared to non-targeting siRNA transfected cells. Values are in fold change and each value is the average of the triplicate samples for each siRNA.
    Figure Legend Snippet: Loss of UBQLN1 induces EMT (a) UBQLN1 loss induces EMT in A549 and H358 cells. A549 and H358 cells were transfected with either with non-targeting siRNA (siNT) or siRNAs targeting UBQLN1 (siU1, siU1-2). After 72 hrs of transfection cells were harvested and analyzed for protein expression using the indicated antibodies. (b) Fluorescence staining for E-cadherin and Vimentin in A549. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting UBQLN1 (siU1 and siU1-2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective E-Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. (c) Cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows. (d) Table indicating the fold change of mRNA following siRNA mediated knockdown of UBQLN1 for EMT-associated genes, as compared to non-targeting siRNA transfected cells. Values are in fold change and each value is the average of the triplicate samples for each siRNA.

    Techniques Used: Transfection, Expressing, Fluorescence, Staining

    15) Product Images from "Enhanced viral-mediated cochlear gene delivery in adult mice by combining canal fenestration with round window membrane inoculation"

    Article Title: Enhanced viral-mediated cochlear gene delivery in adult mice by combining canal fenestration with round window membrane inoculation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21233-z

    Vestibular organs are transduced following the RWM + CF injection. ( a – c ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and CAs of the LSCC and ASCC as indicated. Tissue was harvested 2 weeks after the RWM + CF injection (at P15–16), stained with Alexa Fluor 568-phalloidin (red) for labelling filamentous actin, and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).
    Figure Legend Snippet: Vestibular organs are transduced following the RWM + CF injection. ( a – c ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and CAs of the LSCC and ASCC as indicated. Tissue was harvested 2 weeks after the RWM + CF injection (at P15–16), stained with Alexa Fluor 568-phalloidin (red) for labelling filamentous actin, and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).

    Techniques Used: Injection, Staining

    RWM + CF approach comparing canalostomy of the LSCC and PSCC. ( a ) Inner ear schematic showing the RWM + CF approach with a LSCC canalostomy. ( b ) Representative low magnification images of whole-mount apical turns and high magnification images of the apex, middle and base 2 weeks after injection of AAV2/9 (3.90 × 10 13 vg/ml) delivered at P15–16 using a RWM + CF approach with a canalostomy in LSCC. Cochleae were stained with Myo7a (red) for labelling hair cells and imaged for native eGFP (green). ( c ) Quantification of eGFP-positive IHCs in the apex, middle and base, and cochlear apex 2 weeks following a RWM + CF approach with a canalostomy in LSCC (n = 3). Data are means. ( d ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and the CA of the LSCC and ASCC are shown. Tissue was harvested 2 weeks after injection of AAV2/9 (3.90 × 10 13 vg/ml) delivered at P15–16 using a RWM + CF approach with a canalostomy in LSCC, and stained with Alexa Flour 568-phalloidin (red) for labelling filamentous actin and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately, and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).
    Figure Legend Snippet: RWM + CF approach comparing canalostomy of the LSCC and PSCC. ( a ) Inner ear schematic showing the RWM + CF approach with a LSCC canalostomy. ( b ) Representative low magnification images of whole-mount apical turns and high magnification images of the apex, middle and base 2 weeks after injection of AAV2/9 (3.90 × 10 13 vg/ml) delivered at P15–16 using a RWM + CF approach with a canalostomy in LSCC. Cochleae were stained with Myo7a (red) for labelling hair cells and imaged for native eGFP (green). ( c ) Quantification of eGFP-positive IHCs in the apex, middle and base, and cochlear apex 2 weeks following a RWM + CF approach with a canalostomy in LSCC (n = 3). Data are means. ( d ) Representative images of whole mounts of the CA of the PSCC, saccule, utricle and the CA of the LSCC and ASCC are shown. Tissue was harvested 2 weeks after injection of AAV2/9 (3.90 × 10 13 vg/ml) delivered at P15–16 using a RWM + CF approach with a canalostomy in LSCC, and stained with Alexa Flour 568-phalloidin (red) for labelling filamentous actin and imaged for native eGFP (green). High magnification views of the regions marked with white dotted squares in the PSCC are shown separately, and stained with Myo7a (gray) for labelling hair cells and imaged for native eGFP (green).

    Techniques Used: Injection, Staining

    16) Product Images from "Control of mitochondrial homeostasis by endocytic regulatory proteins"

    Article Title: Control of mitochondrial homeostasis by endocytic regulatory proteins

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.204537

    Depletion of EHD1, rabankyrin-5 or VPS35 does not induce Mfn2 accumulation. HeLa cells were either mock treated, or treated with EHD1, rabankyrin-5 or Vps35 siRNA for 72 h. Depletion efficacy was validated by immunoblotting with antibodies against EHD1, rabankyrin-5 and VPS35 (A; top three panels), and the effect of the siRNA was assessed with antibodies against Mfn2 (A; second panel from the top), Drp1 (A; third panel from the top) and actin (A; bottom panel). (B–G) Densitometric quantification from three separate experiments. * P
    Figure Legend Snippet: Depletion of EHD1, rabankyrin-5 or VPS35 does not induce Mfn2 accumulation. HeLa cells were either mock treated, or treated with EHD1, rabankyrin-5 or Vps35 siRNA for 72 h. Depletion efficacy was validated by immunoblotting with antibodies against EHD1, rabankyrin-5 and VPS35 (A; top three panels), and the effect of the siRNA was assessed with antibodies against Mfn2 (A; second panel from the top), Drp1 (A; third panel from the top) and actin (A; bottom panel). (B–G) Densitometric quantification from three separate experiments. * P

    Techniques Used:

    EHD1 interacts with Mul1. (A) Model for the potential role of EHD1 in regulating mitochondrial dynamics via Mul1. Under normal conditions, the ubiquitin ligase Mul1 is released from an interaction with VPS35 and the retromer components (including EHD1), and relocates to the mitochondrial membrane, where it ubiquitylates Mfn2, inducing its proteasomal degradation and promoting normal mitochondrial fission. Upon EHD1 depletion, Mul1 would be retained in association with VPS35 and the retromer, preventing Mfn2 degradation and thus enhancing mitochondrial membrane fusion. (B) GST pulldown from bovine brain cytosol was performed with GST only, a GST-tagged EH domain of EHD1 (GST–EH1) and GST–EHD1. Eluates were immunoblotted with antibodies against MICAL-L1 (top panel), as a positive interactor with EHD1, and Mul1 (middle panel). GST fusion protein samples were immunoblotted with anti-GST (bottom panel). (C) Co-immunoprecipitation (IP) of proteins from a HeLa cell lysate using anti-Mul1 (αMul1), and immunoblotted with anti-Vps26 and anti-rabankyrin-5 antibodies. 25 kDa immunoglobulin light chains detected by the secondary anti-light chain antibody are indicated in the bottom panel.
    Figure Legend Snippet: EHD1 interacts with Mul1. (A) Model for the potential role of EHD1 in regulating mitochondrial dynamics via Mul1. Under normal conditions, the ubiquitin ligase Mul1 is released from an interaction with VPS35 and the retromer components (including EHD1), and relocates to the mitochondrial membrane, where it ubiquitylates Mfn2, inducing its proteasomal degradation and promoting normal mitochondrial fission. Upon EHD1 depletion, Mul1 would be retained in association with VPS35 and the retromer, preventing Mfn2 degradation and thus enhancing mitochondrial membrane fusion. (B) GST pulldown from bovine brain cytosol was performed with GST only, a GST-tagged EH domain of EHD1 (GST–EH1) and GST–EHD1. Eluates were immunoblotted with antibodies against MICAL-L1 (top panel), as a positive interactor with EHD1, and Mul1 (middle panel). GST fusion protein samples were immunoblotted with anti-GST (bottom panel). (C) Co-immunoprecipitation (IP) of proteins from a HeLa cell lysate using anti-Mul1 (αMul1), and immunoblotted with anti-Vps26 and anti-rabankyrin-5 antibodies. 25 kDa immunoglobulin light chains detected by the secondary anti-light chain antibody are indicated in the bottom panel.

    Techniques Used: Immunoprecipitation

    Depletion of EHD1 and rabankyrin-5 results in reduced and sequestered VPS35, respectively. (A–D) HeLa cells were either mock treated, treated with EHD1 siRNA (A) or treated with rabankyrin-5 (Rank-5) siRNA (C) for 72 h and immunoblotted for VPS35, EHD1, Rank-5 and actin. The asterisk (in A) indicates reduced VPS35 protein levels. (B,D) Quantification of protein levels from three independent experiments. * P
    Figure Legend Snippet: Depletion of EHD1 and rabankyrin-5 results in reduced and sequestered VPS35, respectively. (A–D) HeLa cells were either mock treated, treated with EHD1 siRNA (A) or treated with rabankyrin-5 (Rank-5) siRNA (C) for 72 h and immunoblotted for VPS35, EHD1, Rank-5 and actin. The asterisk (in A) indicates reduced VPS35 protein levels. (B,D) Quantification of protein levels from three independent experiments. * P

    Techniques Used:

    Rabankyrin-5 mediates the interaction between EHD1 and Mul1, and its depletion induces an elongated mitochondrial network similar to that observed upon EHD1 depletion. (A,B) RPE cells were either mock treated (A) or treated with rabankyrin-5 siRNA for 72 h (B) and immunostained for the mitochondrial membrane marker Tom20. (C) The Mito Morphology Macro plugin in ImageJ was used for quantifying mean±s.d. for mitochondrial size, perimeter and circularity in three independent experiments each using 10 cells per treatment. * P
    Figure Legend Snippet: Rabankyrin-5 mediates the interaction between EHD1 and Mul1, and its depletion induces an elongated mitochondrial network similar to that observed upon EHD1 depletion. (A,B) RPE cells were either mock treated (A) or treated with rabankyrin-5 siRNA for 72 h (B) and immunostained for the mitochondrial membrane marker Tom20. (C) The Mito Morphology Macro plugin in ImageJ was used for quantifying mean±s.d. for mitochondrial size, perimeter and circularity in three independent experiments each using 10 cells per treatment. * P

    Techniques Used: Marker

    17) Product Images from "Persistent fibroblast growth factor 23 signalling in the parathyroid glands for secondary hyperparathyroidism in mice with chronic kidney disease"

    Article Title: Persistent fibroblast growth factor 23 signalling in the parathyroid glands for secondary hyperparathyroidism in mice with chronic kidney disease

    Journal: Scientific Reports

    doi: 10.1038/srep40534

    Expression of FGFRs (FGFR1, FGFR2, FGFR3, and FGFR4) and αKlotho in normal and genetically engineered parathyroid glands. A specific gene or genes were conditionally manipulated in the parathyroid glands by mating Fgfr1–3 flox/flox , αKlotho flox/flox , or Fgfr1–4 flox/flox mice with PTH-Cre mice (cKO) or without mating (non-cKO). The mice were treated with heminephrectomy plus a high-phosphate diet (CKD) or not treated (non-CKD) using the protocol described in the Methods section. Paraffin-embedded thyro-parathyroid glands were sectioned and immunostained (red) using an indirect immunofluorescence technique as described in the Methods section. Nuclei were stained with DAPI (blue). Scale bars: 10 μm.
    Figure Legend Snippet: Expression of FGFRs (FGFR1, FGFR2, FGFR3, and FGFR4) and αKlotho in normal and genetically engineered parathyroid glands. A specific gene or genes were conditionally manipulated in the parathyroid glands by mating Fgfr1–3 flox/flox , αKlotho flox/flox , or Fgfr1–4 flox/flox mice with PTH-Cre mice (cKO) or without mating (non-cKO). The mice were treated with heminephrectomy plus a high-phosphate diet (CKD) or not treated (non-CKD) using the protocol described in the Methods section. Paraffin-embedded thyro-parathyroid glands were sectioned and immunostained (red) using an indirect immunofluorescence technique as described in the Methods section. Nuclei were stained with DAPI (blue). Scale bars: 10 μm.

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Staining

    18) Product Images from "Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells"

    Article Title: Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-216

    Cell surface binding of rAbOmpA . The grey shaded region represents the control fluorescent level seen in the cells treated with polyclonal anti-rabbit AbOmpA antibody and Alexa Fluor ® 488-conjugated secondary antibody without the addition of rAbOmpA. The solid line represents the fluorescent level seen in the cells treated with 6 μg/ml of rAbOmpA.
    Figure Legend Snippet: Cell surface binding of rAbOmpA . The grey shaded region represents the control fluorescent level seen in the cells treated with polyclonal anti-rabbit AbOmpA antibody and Alexa Fluor ® 488-conjugated secondary antibody without the addition of rAbOmpA. The solid line represents the fluorescent level seen in the cells treated with 6 μg/ml of rAbOmpA.

    Techniques Used: Binding Assay

    Adherence and invasion of A. baumannii ATCC19606 T and isogenic AbOmpA - mutant in epithelial cells . (A). Adherence of A. baumannii to epithelial cells. NCI-H292 cells were infected with A. baumannii strains at an MOI of 100 for 1 h. Actin was stained with Alexa Fluor ® 488 phalloidin (green). Bacteria were stained with polyclonal anti-rabbit AbOmpA antibody, followed by a secondary antibody Alexa Fluor ® 568 (red). (B). NCI-H292 and HEp-2 cells were infected with A. baumannii at an MOI of 100 for 5 h. Invasion efficiency was expressed as the number of bacteria per well using the gentamicin protection assay. Results represent the mean and standard deviation of three separate experiments on separately days. * P
    Figure Legend Snippet: Adherence and invasion of A. baumannii ATCC19606 T and isogenic AbOmpA - mutant in epithelial cells . (A). Adherence of A. baumannii to epithelial cells. NCI-H292 cells were infected with A. baumannii strains at an MOI of 100 for 1 h. Actin was stained with Alexa Fluor ® 488 phalloidin (green). Bacteria were stained with polyclonal anti-rabbit AbOmpA antibody, followed by a secondary antibody Alexa Fluor ® 568 (red). (B). NCI-H292 and HEp-2 cells were infected with A. baumannii at an MOI of 100 for 5 h. Invasion efficiency was expressed as the number of bacteria per well using the gentamicin protection assay. Results represent the mean and standard deviation of three separate experiments on separately days. * P

    Techniques Used: Mutagenesis, Infection, Staining, Standard Deviation

    Invasion of A. baumannii in epithelial cells . (A) NCI-H292 cells were infected with A. baumannii ATCC 19606 T at an MOI of 100 up to 7 h. The colony-forming units were enumerated to measure the time-course of invasion. The result represents the mean ± standard deviation in duplicate wells and repeated a minimum of three separate times on separate days. (B) NCI-H292, HEp-2, and HeLa cells were infected with A. baumannii strains at an MOI of 100 for 5 h. (C) NCI-H292 cells were infected with A. baumannii 05KA103 at an MOI of 100 for 5 h. Actin was stained with Alexa Fluor ® 488 phalloidin (green). Bacteria were stained with polyclonal anti-rabbit AbOmpA antibody, followed by a secondary antibody Alexa Fluor ® 568 (red). The analytical sectioning was performed from the top to the bottom of the cells. The figure represents a single section of the cells. (D). NCI-H292 cells were infected with A. baumannii ATCC 19606 T at an MOI of 100 for 5 h. Actin filaments have wrap-around-bacteria (white arrow). Red arrow indicates the extracellular bacteria. The figure represents all projection of sections in one picture.
    Figure Legend Snippet: Invasion of A. baumannii in epithelial cells . (A) NCI-H292 cells were infected with A. baumannii ATCC 19606 T at an MOI of 100 up to 7 h. The colony-forming units were enumerated to measure the time-course of invasion. The result represents the mean ± standard deviation in duplicate wells and repeated a minimum of three separate times on separate days. (B) NCI-H292, HEp-2, and HeLa cells were infected with A. baumannii strains at an MOI of 100 for 5 h. (C) NCI-H292 cells were infected with A. baumannii 05KA103 at an MOI of 100 for 5 h. Actin was stained with Alexa Fluor ® 488 phalloidin (green). Bacteria were stained with polyclonal anti-rabbit AbOmpA antibody, followed by a secondary antibody Alexa Fluor ® 568 (red). The analytical sectioning was performed from the top to the bottom of the cells. The figure represents a single section of the cells. (D). NCI-H292 cells were infected with A. baumannii ATCC 19606 T at an MOI of 100 for 5 h. Actin filaments have wrap-around-bacteria (white arrow). Red arrow indicates the extracellular bacteria. The figure represents all projection of sections in one picture.

    Techniques Used: Infection, Standard Deviation, Staining

    19) Product Images from "NTPDase3 and ecto-5′-nucleotidase/CD73 are differentially expressed during mouse bladder cancer progression"

    Article Title: NTPDase3 and ecto-5′-nucleotidase/CD73 are differentially expressed during mouse bladder cancer progression

    Journal: Purinergic Signalling

    doi: 10.1007/s11302-014-9405-8

    a The images correspond to immunofluorescent staining of ecto-5′-NT/CD73 in different times of bladder cancer induction. Cryosections of mouse bladders were labeled with antibody to ecto-5′-NT/CD73 ( red ), Alexa 488 phalloidin to label
    Figure Legend Snippet: a The images correspond to immunofluorescent staining of ecto-5′-NT/CD73 in different times of bladder cancer induction. Cryosections of mouse bladders were labeled with antibody to ecto-5′-NT/CD73 ( red ), Alexa 488 phalloidin to label

    Techniques Used: Staining, Labeling

    a The images correspond to immunofluorescence of NTPDase3 in bladder urothelium at different times of bladder cancer induction. Cryosections of mouse bladders were labeled with antibody to NTPDase3 ( red ), Alexa 488 phalloidin to label actin cytoskeleton
    Figure Legend Snippet: a The images correspond to immunofluorescence of NTPDase3 in bladder urothelium at different times of bladder cancer induction. Cryosections of mouse bladders were labeled with antibody to NTPDase3 ( red ), Alexa 488 phalloidin to label actin cytoskeleton

    Techniques Used: Immunofluorescence, Labeling

    20) Product Images from "Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells"

    Article Title: Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20144322

    Confirmation of the allografted quasi-natural cell block in the recipient body. To confirm allogenicity of the transplanted quasi-natural cell block, gender-specific probes were used to distinguish the male-derived allografted tissue from the recipient female mouse by FISH along with DAPI staining of the nucleus ( A, B ). Green spots indicate male Y chromosome probe in the corresponding female tissue. For intercellular communication capability in the allografted tissue, specimens were stained with specific probes for signaling marker connexin43, in the quasi-natural tissue ( C ) along with native heart tissue ( D ). Immunostaining of the allografted quasi-natural tissue with epithelial linage marker CD31 revealed vascular network assembly as indicated ( E ). Immunostaining of the allografted quasi-natural tissue further showed that most of cells were stained with anti-vimentin, mesodermal lineage marker, but not with anti-cytokeratin ( F ). Images for individual channels (connexin43 with alexa 488 is green, cd31 with alexa 568 is red, vimentin with alexa 488 is green, cytokeratin with alexa 568 is red) are shown on the left, and main panels show the merged image containing all channels plus DIC. The cells nuclei were visualized with DAPI (blue). Scale bar: ( A, C, D, E and F ) 20 μm; ( B ) 100 μm.
    Figure Legend Snippet: Confirmation of the allografted quasi-natural cell block in the recipient body. To confirm allogenicity of the transplanted quasi-natural cell block, gender-specific probes were used to distinguish the male-derived allografted tissue from the recipient female mouse by FISH along with DAPI staining of the nucleus ( A, B ). Green spots indicate male Y chromosome probe in the corresponding female tissue. For intercellular communication capability in the allografted tissue, specimens were stained with specific probes for signaling marker connexin43, in the quasi-natural tissue ( C ) along with native heart tissue ( D ). Immunostaining of the allografted quasi-natural tissue with epithelial linage marker CD31 revealed vascular network assembly as indicated ( E ). Immunostaining of the allografted quasi-natural tissue further showed that most of cells were stained with anti-vimentin, mesodermal lineage marker, but not with anti-cytokeratin ( F ). Images for individual channels (connexin43 with alexa 488 is green, cd31 with alexa 568 is red, vimentin with alexa 488 is green, cytokeratin with alexa 568 is red) are shown on the left, and main panels show the merged image containing all channels plus DIC. The cells nuclei were visualized with DAPI (blue). Scale bar: ( A, C, D, E and F ) 20 μm; ( B ) 100 μm.

    Techniques Used: Blocking Assay, Derivative Assay, Fluorescence In Situ Hybridization, Staining, Marker, Immunostaining

    21) Product Images from "Miniature- and Multiple-Eyespot Loci in Chlamydomonas reinhardtii Define New Modulators of Eyespot Photoreception and Assembly"

    Article Title: Miniature- and Multiple-Eyespot Loci in Chlamydomonas reinhardtii Define New Modulators of Eyespot Photoreception and Assembly

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.111.000679

    ChR1 photoreceptor localization and eyespot layers are altered in min1 but not min2 . (A–D) Combined immunofluorescence micrographs of fixed cells stained for channelrhodopsin-1 (ChR1, magenta) and acetylated α-tubulin (AcTub, green). (A) Wild-type cell with a ChR1 patch associated with the D4 microtubule rootlet. (B) ChR1 staining in min1 cells appears as multiple, distinct spots or stripes along the D4 rootlet, occasionally appearing in off-rootlet spots. (C) The shape and position of the ChR1 patch on the D4 rootlet are maintained in min2 mutant cells. (D) min1 min2 cells, showing ChR1 staining in multiple spots along the D4 rootlet. (E–F) Combined immunofluorescence micrographs of fixed cells stained for the pigment granule marker EYE3 (red), ChR1 (blue), and AcTub (green). (E) Pigment granules (arrow) are not apposed to the plasma membrane-localized photoreceptor spots in photoautotrophically grown min1 cells. (F) Organization of eyespot layers is unaffected in min2 cells, with ChR1 directly overlaying EYE3 staining (inset). Scale bars, 5 μm.
    Figure Legend Snippet: ChR1 photoreceptor localization and eyespot layers are altered in min1 but not min2 . (A–D) Combined immunofluorescence micrographs of fixed cells stained for channelrhodopsin-1 (ChR1, magenta) and acetylated α-tubulin (AcTub, green). (A) Wild-type cell with a ChR1 patch associated with the D4 microtubule rootlet. (B) ChR1 staining in min1 cells appears as multiple, distinct spots or stripes along the D4 rootlet, occasionally appearing in off-rootlet spots. (C) The shape and position of the ChR1 patch on the D4 rootlet are maintained in min2 mutant cells. (D) min1 min2 cells, showing ChR1 staining in multiple spots along the D4 rootlet. (E–F) Combined immunofluorescence micrographs of fixed cells stained for the pigment granule marker EYE3 (red), ChR1 (blue), and AcTub (green). (E) Pigment granules (arrow) are not apposed to the plasma membrane-localized photoreceptor spots in photoautotrophically grown min1 cells. (F) Organization of eyespot layers is unaffected in min2 cells, with ChR1 directly overlaying EYE3 staining (inset). Scale bars, 5 μm.

    Techniques Used: Immunofluorescence, Staining, Mutagenesis, Marker

    Quantification of the extent of EYE2, EYE3, and ChR1 copositioning in mlt1 and mlt2 cells. Percentage of single or copositioned spots per total spots scored is shown. Copositioned spots of combinations of EYE2, EYE3, and ChR1 are markedly more prevalent in mlt1 cells compared with mlt2 cells. Conversely, mlt2 cells have a greater proportion of single EYE2, EYE3, or ChR1 spots not copositioned with other eyespot proteins. Copositioning of EYE3 with ChR1 was never observed without EYE2.
    Figure Legend Snippet: Quantification of the extent of EYE2, EYE3, and ChR1 copositioning in mlt1 and mlt2 cells. Percentage of single or copositioned spots per total spots scored is shown. Copositioned spots of combinations of EYE2, EYE3, and ChR1 are markedly more prevalent in mlt1 cells compared with mlt2 cells. Conversely, mlt2 cells have a greater proportion of single EYE2, EYE3, or ChR1 spots not copositioned with other eyespot proteins. Copositioning of EYE3 with ChR1 was never observed without EYE2.

    Techniques Used:

    Eyespots are disorganized in mlt1 and mlt2 mutant cells. (A, B) Combined immunofluorescence micrographs of individual cells stained for ChR1 (magenta) and AcTub (green). (A) mlt1 cells have ChR1 patches in either hemisphere of the cell that are often clustered around the anterior pole and associated with acetylated rootlets. Arrow indicates ChR1 patch not associated with a rootlet. (B) mlt2 cell with multiple ChR1 patches associated with microtubule rootlets. (C) Wild-type cell showing layered arrangement of EYE2 (green), EYE3 (blue), and ChR1 (red) in the eyespot (inset). (D–F) EYE2, EYE3, and ChR1 positioning is dramatically disrupted in asynchronous stationary-phase populations of mlt1 and mlt2 cells. Combined immunofluorescence micrographs of fixed cells stained for EYE2 (green), EYE3 (blue), and ChR1 (red). a, single EYE2 spot; b:, single ChR1 spot; c, single EYE3 spot; d, EYE2/EYE3 copositioned spot; e, EYE2/ChR1 copositioned spot; f, EYE2/EYE3/ChR1 copositioned spot. (D) Z-projections of combined immunofluorescence micrographs of an individual mlt2 cell illustrating positioning of EYE2, EYE3, and ChR1 spots. (E) Z-projection of combined immunofluorescence micrographs of a mlt1 field stained for EYE2 (green), EYE3 (blue), and ChR1 (red). Various combinations of single and copositioned spots are observed (arrows). (F) Z-projection of combined immunofluorescence micrographs of a mlt2 field. Various spot-positioning combinations are again observed, with single spots predominating. Scale bars, 5 µm.
    Figure Legend Snippet: Eyespots are disorganized in mlt1 and mlt2 mutant cells. (A, B) Combined immunofluorescence micrographs of individual cells stained for ChR1 (magenta) and AcTub (green). (A) mlt1 cells have ChR1 patches in either hemisphere of the cell that are often clustered around the anterior pole and associated with acetylated rootlets. Arrow indicates ChR1 patch not associated with a rootlet. (B) mlt2 cell with multiple ChR1 patches associated with microtubule rootlets. (C) Wild-type cell showing layered arrangement of EYE2 (green), EYE3 (blue), and ChR1 (red) in the eyespot (inset). (D–F) EYE2, EYE3, and ChR1 positioning is dramatically disrupted in asynchronous stationary-phase populations of mlt1 and mlt2 cells. Combined immunofluorescence micrographs of fixed cells stained for EYE2 (green), EYE3 (blue), and ChR1 (red). a, single EYE2 spot; b:, single ChR1 spot; c, single EYE3 spot; d, EYE2/EYE3 copositioned spot; e, EYE2/ChR1 copositioned spot; f, EYE2/EYE3/ChR1 copositioned spot. (D) Z-projections of combined immunofluorescence micrographs of an individual mlt2 cell illustrating positioning of EYE2, EYE3, and ChR1 spots. (E) Z-projection of combined immunofluorescence micrographs of a mlt1 field stained for EYE2 (green), EYE3 (blue), and ChR1 (red). Various combinations of single and copositioned spots are observed (arrows). (F) Z-projection of combined immunofluorescence micrographs of a mlt2 field. Various spot-positioning combinations are again observed, with single spots predominating. Scale bars, 5 µm.

    Techniques Used: Mutagenesis, Immunofluorescence, Staining

    22) Product Images from "Targeting Swine Leukocyte Antigen Class I Molecules for Proteasomal Degradation by the nsp1α Replicase Protein of the Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06"

    Article Title: Targeting Swine Leukocyte Antigen Class I Molecules for Proteasomal Degradation by the nsp1α Replicase Protein of the Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06

    Journal: Journal of Virology

    doi: 10.1128/JVI.02307-15

    Downregulation of SLA-I cell surface accumulation of PAMs by HP-PRRSV. (A) Effect of PRRSV on SLA-I cell surface accumulation. PAMs were mock infected or infected with HP-PRRSV JXwn06 or UV-irradiated JXwn06 or HB-1/3.9 at an MOI of 1 or 0.1. At the indicated time points, the cells were harvested and subjected to FACS analysis with mouse monoclonal antibody against SLA-I (JM1E3). (B) PRRSV- or mock-inoculated PAMs were incubated with saturating amounts of JM1E3 at 4°C for 1 h before being washed three times with ice-cold PBS to remove unbound MAbs. The cells were then incubated at 37°C to promote endocytosis. At different times postinfection, the cells were collected and subjected to FACS analysis with FITC-conjugated goat anti-mouse IgG. (C) PRRSV- or mock-infected PAMs were incubated with saturating amounts of unlabeled MAb JM1E3 to SLA-I at 4°C for 1 h and then at 37°C for various times before being subjected to FACS analysis with FITC-conjugated JM1E3. The trend curves were fitted based on the MFIs, and the data shown are means and standard deviations of results from three independent experiments (*, P
    Figure Legend Snippet: Downregulation of SLA-I cell surface accumulation of PAMs by HP-PRRSV. (A) Effect of PRRSV on SLA-I cell surface accumulation. PAMs were mock infected or infected with HP-PRRSV JXwn06 or UV-irradiated JXwn06 or HB-1/3.9 at an MOI of 1 or 0.1. At the indicated time points, the cells were harvested and subjected to FACS analysis with mouse monoclonal antibody against SLA-I (JM1E3). (B) PRRSV- or mock-inoculated PAMs were incubated with saturating amounts of JM1E3 at 4°C for 1 h before being washed three times with ice-cold PBS to remove unbound MAbs. The cells were then incubated at 37°C to promote endocytosis. At different times postinfection, the cells were collected and subjected to FACS analysis with FITC-conjugated goat anti-mouse IgG. (C) PRRSV- or mock-infected PAMs were incubated with saturating amounts of unlabeled MAb JM1E3 to SLA-I at 4°C for 1 h and then at 37°C for various times before being subjected to FACS analysis with FITC-conjugated JM1E3. The trend curves were fitted based on the MFIs, and the data shown are means and standard deviations of results from three independent experiments (*, P

    Techniques Used: Infection, Irradiation, FACS, Incubation

    23) Product Images from "The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation"

    Article Title: The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation

    Journal: Infection and Immunity

    doi: 10.1128/IAI.03118-14

    Raft-like microdomains in Giardia trophozoites. Trophozoites were labeled with Alexa Fluor 488-conjugated CTXB (image a, green) and GM1 antibody (image b, red). Labeling of plasma membranes, the ventral disc, and the flagella of trophozoites is visible. FAST Dil oil labels the cytoplasmic and nuclear lipids of trophozoites (image c). DAPI-stained nuclei are also noticeable. N, nucleus; PM, plasma membrane; F, flagella; VD, ventral disc; ab, antibody. Bars, 5 μm. (B) 3D representation. The image of CTXB-labeled Giardia trophozoites was captured using Zen 2009 software. z-stacks were acquired, and a 3D model was reconstructed from the 12 optical sections of the z-stacks with a slice thickness of 0.37 μm each.
    Figure Legend Snippet: Raft-like microdomains in Giardia trophozoites. Trophozoites were labeled with Alexa Fluor 488-conjugated CTXB (image a, green) and GM1 antibody (image b, red). Labeling of plasma membranes, the ventral disc, and the flagella of trophozoites is visible. FAST Dil oil labels the cytoplasmic and nuclear lipids of trophozoites (image c). DAPI-stained nuclei are also noticeable. N, nucleus; PM, plasma membrane; F, flagella; VD, ventral disc; ab, antibody. Bars, 5 μm. (B) 3D representation. The image of CTXB-labeled Giardia trophozoites was captured using Zen 2009 software. z-stacks were acquired, and a 3D model was reconstructed from the 12 optical sections of the z-stacks with a slice thickness of 0.37 μm each.

    Techniques Used: Labeling, Staining, Software

    (A) Expression of raft-like microdomains by encysting Giardia cultured in 5% DFBS-containing medium. The cells were labeled with Alexa Fluor 488-conjugated CTXB and DAPI and viewed under a confocal microscope. (Image a) Nonencysting trophozoites; (image b) encysting cells at 10 h p.i. of encystation; (image c) encysting cells at 18 h p.i. of encystation; (image d) water-resistant cysts. (B) ESV biogenesis and expression of trophozoite proteins. Encysting cells at 10 h p.i. of encystation (control and inhibitor treated) were labeled with trophozoite (green) and cyst (red) antibodies as described in Materials and Methods. (Image a) Labeling of encysting cells at 10 h p.i. of encystation in medium supplemented with ABS; (image b) encysting trophozoites at 10 h p.i. of encystation differentiated in medium containing DFBS; (image c) nystatin (27 μM) treatment; (image d) filipin III (7.6 μM) treatment; (image e) oseltamivir (20 μM) treatment. (C) Induction of encystation in DFBS-supplemented medium affects cyst production. Control and inhibitor-treated trophozoites were allowed to encyst for 18 h before labeling with cyst (red) and trophozoite (green) antibodies. (Image a) Cyst antibody-labeled water-resistant cysts produced in ABS-supplemented medium; (image b) cysts generated in ABS-supplemented medium; (image c) nystatin (27 μM) treatment; image d, filipin III (7.6 μM) treatment; image e, oseltamivir (20 μM) treatment. N, nucleus; ESV, encystation-specific vesicle; PM, plasma membrane; N, nucleus; VD, ventral disc; F, flagella; CW, cyst wall; NCW, nascent cyst wall; CL, cyst-like structure; TL, trophozoite-like structure. Bars, 5 μm. (D) Quantitative estimates of water-resistant cells expressing trophozoite proteins or cyst proteins. For quantification, 125 cells from 6 randomly selected fields from 2 separate experiments were counted. Data were analyzed by a one-way ANOVA followed by the Holm-Šídák method. **, P
    Figure Legend Snippet: (A) Expression of raft-like microdomains by encysting Giardia cultured in 5% DFBS-containing medium. The cells were labeled with Alexa Fluor 488-conjugated CTXB and DAPI and viewed under a confocal microscope. (Image a) Nonencysting trophozoites; (image b) encysting cells at 10 h p.i. of encystation; (image c) encysting cells at 18 h p.i. of encystation; (image d) water-resistant cysts. (B) ESV biogenesis and expression of trophozoite proteins. Encysting cells at 10 h p.i. of encystation (control and inhibitor treated) were labeled with trophozoite (green) and cyst (red) antibodies as described in Materials and Methods. (Image a) Labeling of encysting cells at 10 h p.i. of encystation in medium supplemented with ABS; (image b) encysting trophozoites at 10 h p.i. of encystation differentiated in medium containing DFBS; (image c) nystatin (27 μM) treatment; (image d) filipin III (7.6 μM) treatment; (image e) oseltamivir (20 μM) treatment. (C) Induction of encystation in DFBS-supplemented medium affects cyst production. Control and inhibitor-treated trophozoites were allowed to encyst for 18 h before labeling with cyst (red) and trophozoite (green) antibodies. (Image a) Cyst antibody-labeled water-resistant cysts produced in ABS-supplemented medium; (image b) cysts generated in ABS-supplemented medium; (image c) nystatin (27 μM) treatment; image d, filipin III (7.6 μM) treatment; image e, oseltamivir (20 μM) treatment. N, nucleus; ESV, encystation-specific vesicle; PM, plasma membrane; N, nucleus; VD, ventral disc; F, flagella; CW, cyst wall; NCW, nascent cyst wall; CL, cyst-like structure; TL, trophozoite-like structure. Bars, 5 μm. (D) Quantitative estimates of water-resistant cells expressing trophozoite proteins or cyst proteins. For quantification, 125 cells from 6 randomly selected fields from 2 separate experiments were counted. Data were analyzed by a one-way ANOVA followed by the Holm-Šídák method. **, P

    Techniques Used: Expressing, Cell Culture, Labeling, Microscopy, Produced, Generated

    24) Product Images from "The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans"

    Article Title: The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-06-0622

    Depletion of endogenous GCC88 by an inducible shRNA. HeLa cells (clone A8) stably expressing a tetracycline on inducible shRNA to GCC88 (tet R GCC88KD A8) were either untreated (control) or treated with 10 or 100 ng/ml doxycycline (Dox) for 96 h, and monolayers were fixed with 4% paraformaldehyde. (A) Endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 488-conjugated anti-rabbit IgG. (B) HeLa A8 cells were incubated with 10 ng/ml doxycycline for 96 h, lysed in SDS-PAGE reducing buffer, and then extracts were subjected to SDS-PAGE on a 7.5% polyacrylamide gel. Proteins were transfer to a polyvinylidene difluoride membrane and probed with rabbit anti-GCC88 antibodies using a chemiluminescence detection system. The membrane were then stripped and reprobed with anti-α-tubulin, followed by anti-GCC185 and anti-golgin-97 antibodies. Bar, 10 μm.
    Figure Legend Snippet: Depletion of endogenous GCC88 by an inducible shRNA. HeLa cells (clone A8) stably expressing a tetracycline on inducible shRNA to GCC88 (tet R GCC88KD A8) were either untreated (control) or treated with 10 or 100 ng/ml doxycycline (Dox) for 96 h, and monolayers were fixed with 4% paraformaldehyde. (A) Endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 488-conjugated anti-rabbit IgG. (B) HeLa A8 cells were incubated with 10 ng/ml doxycycline for 96 h, lysed in SDS-PAGE reducing buffer, and then extracts were subjected to SDS-PAGE on a 7.5% polyacrylamide gel. Proteins were transfer to a polyvinylidene difluoride membrane and probed with rabbit anti-GCC88 antibodies using a chemiluminescence detection system. The membrane were then stripped and reprobed with anti-α-tubulin, followed by anti-GCC185 and anti-golgin-97 antibodies. Bar, 10 μm.

    Techniques Used: shRNA, Stable Transfection, Expressing, Incubation, SDS Page

    Defect in TGN38 recycling is rescued by expression of wild-type syntaxin 6. HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and cotransfected with TGN38 and either GFP-syntaxin6 FL (GFP-Syn6 FL ) (A), GFP-syntaxin6 cyto (GFP-Syn6 cyto ) (B), or cherry-syntaxin 16 FL (Cherry-Syn16 FL ) (C) for 24 h before the internalization assay. Monolayers were incubated with monoclonal mouse anti-TGN38 antibodies for 30 min on ice, washed in PBS, and then incubated at 37°C in serum-free media 120 min to internalize the antibody–TGN38 complex. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa 568-conjugated anti-mouse IgG for 60 min. Endogenous GCC88 was stained with rabbit anti-GCC88 antibodies, followed by Alexa 647-conjugated anti-rabbit IgG. Cells with no GCC88 staining and perinuclear level of syntaxin 6 or syntaxin 16 expression were analyzed (n = 15, in duplicate). Bars, 10 μm.
    Figure Legend Snippet: Defect in TGN38 recycling is rescued by expression of wild-type syntaxin 6. HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and cotransfected with TGN38 and either GFP-syntaxin6 FL (GFP-Syn6 FL ) (A), GFP-syntaxin6 cyto (GFP-Syn6 cyto ) (B), or cherry-syntaxin 16 FL (Cherry-Syn16 FL ) (C) for 24 h before the internalization assay. Monolayers were incubated with monoclonal mouse anti-TGN38 antibodies for 30 min on ice, washed in PBS, and then incubated at 37°C in serum-free media 120 min to internalize the antibody–TGN38 complex. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa 568-conjugated anti-mouse IgG for 60 min. Endogenous GCC88 was stained with rabbit anti-GCC88 antibodies, followed by Alexa 647-conjugated anti-rabbit IgG. Cells with no GCC88 staining and perinuclear level of syntaxin 6 or syntaxin 16 expression were analyzed (n = 15, in duplicate). Bars, 10 μm.

    Techniques Used: Expressing, Incubation, Staining

    Shiga toxin is transported efficiently to the Golgi in GCC88-depleted cells. HeLa A8 cells were either untreated (control) (A) or incubated with 10 ng/ml doxycycline for 96 h (GCC88 depleted) (B). Monolayers were incubated with Cy3-conjugated STx-B for 45 min on ice, and then they were either fixed immediately (0 min) or incubated at 37°C for either 20 or 60 min, followed by fixation in 4% paraformaldehyde. Cells were stained with monoclonal antibodies to GM130 followed by Alexa-conjugated anti-mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Shiga toxin is transported efficiently to the Golgi in GCC88-depleted cells. HeLa A8 cells were either untreated (control) (A) or incubated with 10 ng/ml doxycycline for 96 h (GCC88 depleted) (B). Monolayers were incubated with Cy3-conjugated STx-B for 45 min on ice, and then they were either fixed immediately (0 min) or incubated at 37°C for either 20 or 60 min, followed by fixation in 4% paraformaldehyde. Cells were stained with monoclonal antibodies to GM130 followed by Alexa-conjugated anti-mouse IgG. Bars, 10 μm.

    Techniques Used: Incubation, Staining

    Anterograde transport of E-cadherin in GCC88-depleted cells is unaffected. HeLa A8 cells were either untreated (control) or incubated with 10 ng/ml Dox for 72 h and then transfected with Ecad-GFP for 24 h before staining. (A) Monolayers were fixed with 4% paraformaldehyde, permeabilized, and endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 568-conjugated anti-rabbit IgG and Ecad-GFP by GFP fluorescence. (B) Monolayers were fixed and stained with monoclonal anti-Ecad antibodies followed by Alexa 568-conjugated goat ant-mouse IgG. Fixed monolayers were then permeabilized and stained with rabbit anti-GCC88 antibodies followed by Alexa 647-conjugated goat anti-rabbit IgG. Bars, 10 μm.
    Figure Legend Snippet: Anterograde transport of E-cadherin in GCC88-depleted cells is unaffected. HeLa A8 cells were either untreated (control) or incubated with 10 ng/ml Dox for 72 h and then transfected with Ecad-GFP for 24 h before staining. (A) Monolayers were fixed with 4% paraformaldehyde, permeabilized, and endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 568-conjugated anti-rabbit IgG and Ecad-GFP by GFP fluorescence. (B) Monolayers were fixed and stained with monoclonal anti-Ecad antibodies followed by Alexa 568-conjugated goat ant-mouse IgG. Fixed monolayers were then permeabilized and stained with rabbit anti-GCC88 antibodies followed by Alexa 647-conjugated goat anti-rabbit IgG. Bars, 10 μm.

    Techniques Used: Incubation, Transfection, Staining, Fluorescence

    Syntaxin 6 depletion impairs TGN38, but not Shiga toxin, trafficking to the Golgi apparatus. (A) HeLa cells were transfected with syntaxin 6 siRNA for 72 h, fixed in 4% paraformaldehyde, saponin permeablized, and stained with monoclonal anti-syntaxin 6 antibodies followed by Alexa-conjugated mouse IgG. (B) HeLa cells transfected with siRNA as described above for 48 h, and then they were transfected a second time with CFP-TGN38 for a further 24 h. Monolayers were then incubated with monoclonal mouse anti-TGN38 antibodies on ice for 30 min, washed in PBS, and incubated in serum-free media at 37°C for 120 min. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa-conjugated anti-mouse IgG. Endogenous GMAP-210 was stained with rabbit anti-GMAP-210, followed by Alexa-conjugated anti-rabbit IgG. C) HeLa cells were transfected with syntaxin 6 siRNA for 72 h and incubated with Cy3-conjugated STx-B for 45 min on ice and then either fixed immediately (0 min) or incubated at 37°C for 60 min followed by fixation. Cells were stained with monoclonal antibodies to GM130 followed by Alexa 647-conjugated mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Syntaxin 6 depletion impairs TGN38, but not Shiga toxin, trafficking to the Golgi apparatus. (A) HeLa cells were transfected with syntaxin 6 siRNA for 72 h, fixed in 4% paraformaldehyde, saponin permeablized, and stained with monoclonal anti-syntaxin 6 antibodies followed by Alexa-conjugated mouse IgG. (B) HeLa cells transfected with siRNA as described above for 48 h, and then they were transfected a second time with CFP-TGN38 for a further 24 h. Monolayers were then incubated with monoclonal mouse anti-TGN38 antibodies on ice for 30 min, washed in PBS, and incubated in serum-free media at 37°C for 120 min. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa-conjugated anti-mouse IgG. Endogenous GMAP-210 was stained with rabbit anti-GMAP-210, followed by Alexa-conjugated anti-rabbit IgG. C) HeLa cells were transfected with syntaxin 6 siRNA for 72 h and incubated with Cy3-conjugated STx-B for 45 min on ice and then either fixed immediately (0 min) or incubated at 37°C for 60 min followed by fixation. Cells were stained with monoclonal antibodies to GM130 followed by Alexa 647-conjugated mouse IgG. Bars, 10 μm.

    Techniques Used: Transfection, Staining, Incubation

    Depletion of GCC88 results in mislocalization of syntaxin 6. (A–E) HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and then fixed in 4% paraformaldehyde and permeablized. Fixed monolayers were stained for endogenous VAMP4 (A), VAMP3 (B), Vti1a (C), syntaxin 6 (D), and syntaxin 16 (E), with rabbit polyclonal antibodies to VAMP4, VAMP3, and syntaxin 16, respectively, followed by Alexa 568-conjugated rabbit IgG and mouse monoclonal antibodies to vti1a and syntaxin 6, respectively, followed by Alexa 568-conjugated anti-mouse IgG. (F) Untransfected HeLa cells (control) or HeLa cells transfected with p230 siRNA (p230 depleted) for 72 h were fixed in 4% paraformaldehyde and saponin permeabilized. Endogenous p230 was stained with human anti-p230 antibodies followed by FITC-conjugated anti-human IgG and syntaxin 6 stained with monoclonal mouse anti-syntaxin 6, followed by Alexa 568-conjugated anti-mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Depletion of GCC88 results in mislocalization of syntaxin 6. (A–E) HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and then fixed in 4% paraformaldehyde and permeablized. Fixed monolayers were stained for endogenous VAMP4 (A), VAMP3 (B), Vti1a (C), syntaxin 6 (D), and syntaxin 16 (E), with rabbit polyclonal antibodies to VAMP4, VAMP3, and syntaxin 16, respectively, followed by Alexa 568-conjugated rabbit IgG and mouse monoclonal antibodies to vti1a and syntaxin 6, respectively, followed by Alexa 568-conjugated anti-mouse IgG. (F) Untransfected HeLa cells (control) or HeLa cells transfected with p230 siRNA (p230 depleted) for 72 h were fixed in 4% paraformaldehyde and saponin permeabilized. Endogenous p230 was stained with human anti-p230 antibodies followed by FITC-conjugated anti-human IgG and syntaxin 6 stained with monoclonal mouse anti-syntaxin 6, followed by Alexa 568-conjugated anti-mouse IgG. Bars, 10 μm.

    Techniques Used: Incubation, Staining, Transfection

    25) Product Images from "Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells"

    Article Title: Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells

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

    doi: 10.4049/jimmunol.1002835

    Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p
    Figure Legend Snippet: Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, Flow Cytometry

    Knockdown of EC nectin-2 and PVR inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or siRNA targeting nectin-2 (nectin-2 siRNA), PVR (PVR siRNA), or both (nectin-2+PVR siRNA), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–nectin-2 or anti-PVR (thick lines). B , Graphs display data combined from three separate experiments using T cells from different donors. *** p
    Figure Legend Snippet: Knockdown of EC nectin-2 and PVR inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or siRNA targeting nectin-2 (nectin-2 siRNA), PVR (PVR siRNA), or both (nectin-2+PVR siRNA), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–nectin-2 or anti-PVR (thick lines). B , Graphs display data combined from three separate experiments using T cells from different donors. *** p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining

    26) Product Images from "Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells"

    Article Title: Identification of Endothelial Cell Junctional Proteins and Lymphocyte Receptors Involved in Transendothelial Migration of Human Effector Memory CD4+ T Cells

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

    doi: 10.4049/jimmunol.1002835

    Knockdown of EC JAM-1 inhibits chemokine-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or two different siRNAs targeting JAM-1 (JAM-1 siRNA-5 and JAM-1 siRNA-6), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen, and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–JAM-1 (thick lines) demonstrating effective knockdown. B , Left lower panel (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Right lower panel on (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of three (VB2 − ) and two (VB2 + ) separate experiments using T cells from different donors. * p
    Figure Legend Snippet: Knockdown of EC JAM-1 inhibits chemokine-dependent TEM of EM CD4 + T cells. CIITA HDMECs were transfected with control siRNA or two different siRNAs targeting JAM-1 (JAM-1 siRNA-5 and JAM-1 siRNA-6), treated with TNF, either harvested for FACS analysis ( A ) or overlaid with TSST-1 superantigen, and used in flow TEM assays with EM CD4 + T cells ( B ). A , Histograms show FACS analysis of cells stained with isotype control IgG (thin line) or anti–JAM-1 (thick lines) demonstrating effective knockdown. B , Left lower panel (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Right lower panel on (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of three (VB2 − ) and two (VB2 + ) separate experiments using T cells from different donors. * p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining

    Blocking of T cell LFA-1 but not Mac-1 inhibits TCR-dependent TEM of EM CD4 + T cells. A , Mac-1 is expressed on EM CD4 + T cells. Contour plots of FACS analysis of CD45RA − CD4 + T cells stained with isotype-matched control PE- and FITC-conjugated IgG ( left plot ) or PE-conjugated anti-CD11b and FITC-conjugated anti-CCR7 ( right plot ). Note that there is a significant proportion of CD11b + cells in the CCR7 low (i.e., EM) population. B , TEM assays. EM CD4 + T cells were preincubated with isotype control IgG (control), anti–LFA-1 (LFA-1), or anti–Mac-1 (Mac-1) blocking mAb 30 min prior to flow TEM on TNF-treated CIITA-transduced HDMECs overlaid with TSST-1. Panel on the left (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Panel on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data pooled from three separate experiments using T cells isolated from three different donors. *** p
    Figure Legend Snippet: Blocking of T cell LFA-1 but not Mac-1 inhibits TCR-dependent TEM of EM CD4 + T cells. A , Mac-1 is expressed on EM CD4 + T cells. Contour plots of FACS analysis of CD45RA − CD4 + T cells stained with isotype-matched control PE- and FITC-conjugated IgG ( left plot ) or PE-conjugated anti-CD11b and FITC-conjugated anti-CCR7 ( right plot ). Note that there is a significant proportion of CD11b + cells in the CCR7 low (i.e., EM) population. B , TEM assays. EM CD4 + T cells were preincubated with isotype control IgG (control), anti–LFA-1 (LFA-1), or anti–Mac-1 (Mac-1) blocking mAb 30 min prior to flow TEM on TNF-treated CIITA-transduced HDMECs overlaid with TSST-1. Panel on the left (VB2 − ) shows TEM of Vβ2TCR − cells at 15 min. Panel on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data pooled from three separate experiments using T cells isolated from three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, FACS, Staining, Flow Cytometry, Isolation

    Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p
    Figure Legend Snippet: Blocking of EC nectin-2 and/or PVR inhibits TCR-dependent TEM of EM CD4 + T cells. TNF-treated CIITA-transduced HDMECs overlaid with TSST-1 were preincubated with isotype control (control), anti–nectin-2 (nectin-2), anti-PVR (PVR), and both blocking Abs (nectin-2+PVR) prior to flow TEM. Panel on the left shows TEM of Vβ2TCR − cells at 15 min. Panel on the right shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative of eight different experiments, testing each condition with T cells from at least three different donors. *** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, Flow Cytometry

    Blocking of T cell DNAM-1 and/or Tactile inhibits TCR-dependent TEM of EM CD4 + T cells. A , Contour plots of FACS analysis of total CD4 + T cells stained with FITC-conjugated IgG (IgG–FITC) and PE-conjugated IgG (IgG–PE, upper left ) or FITC-conjugated anti-CCR7 (CCR7–FITC) and PE-conjugated anti–DNAM-1 (DNAM-1–PE, lower left ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed IgG (IgG-647, upper right ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed anti-Tactile (Tactile-647, lower right ). Note the distinct population of cells that are DNAM-1 high, CCR7 low or Tactile high, CCR7 low in the middle , left , and right plots , respectively. Lower histograms show overlays of the isotype-matched control IgG and anti–DNAM-1 ( left ) or anti-Tactile ( right ) plots. B , EM CD4 + T cells were preincubated with isotype-matched control IgG (control), anti–DNAM-1 (DNAM-1), anti-Tactile (Tactile), and both anti–DNAM-1 and anti-Tactile blocking mAbs (DNAM-1+Tactile) prior to flow TEM. Left panel shows TEM of Vβ2TCR − cells at 15 min. Right panel shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) and four (VB2 + ) separate experiments using T cells isolated from different donors. ** p
    Figure Legend Snippet: Blocking of T cell DNAM-1 and/or Tactile inhibits TCR-dependent TEM of EM CD4 + T cells. A , Contour plots of FACS analysis of total CD4 + T cells stained with FITC-conjugated IgG (IgG–FITC) and PE-conjugated IgG (IgG–PE, upper left ) or FITC-conjugated anti-CCR7 (CCR7–FITC) and PE-conjugated anti–DNAM-1 (DNAM-1–PE, lower left ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed IgG (IgG-647, upper right ) or FITC-conjugated anti-CCR7 and Alexa Fluor 647-complexed anti-Tactile (Tactile-647, lower right ). Note the distinct population of cells that are DNAM-1 high, CCR7 low or Tactile high, CCR7 low in the middle , left , and right plots , respectively. Lower histograms show overlays of the isotype-matched control IgG and anti–DNAM-1 ( left ) or anti-Tactile ( right ) plots. B , EM CD4 + T cells were preincubated with isotype-matched control IgG (control), anti–DNAM-1 (DNAM-1), anti-Tactile (Tactile), and both anti–DNAM-1 and anti-Tactile blocking mAbs (DNAM-1+Tactile) prior to flow TEM. Left panel shows TEM of Vβ2TCR − cells at 15 min. Right panel shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) and four (VB2 + ) separate experiments using T cells isolated from different donors. ** p

    Techniques Used: Blocking Assay, Transmission Electron Microscopy, FACS, Staining, Flow Cytometry, Isolation

    Knockdown of EC CD99 inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMEC were transfected with control siRNA or two different siRNAs targeting CD99 (CD99 siRNA-2 and CD99 siRNA-5), treated with TNF, analyzed by FACS ( A ) or overlaid with TSST-1 superantigen (recognized by those T cells with the germline-encoded Vβ2 segment in the TCR), and used in flow TEM assays with EM CD4 + T cells ( B ). A , FACS plots of HDMECs stained with isotype-matched control IgG (thin lines) or anti-CD99 or -CD31 (thick lines in left panels and right panels , respectively) demonstrating knockdown of CD99 without reduction of CD31 expression. B , TEM assays. Graph on the left (VB2 − ) shows TEM of Vβ2TCR − cells (i.e., those T cells with TCR that are not activated by TSST-1) at 15 min. Graph on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) or three (VB2 + ) separate experiments using T cells from different donors. *** p
    Figure Legend Snippet: Knockdown of EC CD99 inhibits TCR-dependent TEM of EM CD4 + T cells. CIITA HDMEC were transfected with control siRNA or two different siRNAs targeting CD99 (CD99 siRNA-2 and CD99 siRNA-5), treated with TNF, analyzed by FACS ( A ) or overlaid with TSST-1 superantigen (recognized by those T cells with the germline-encoded Vβ2 segment in the TCR), and used in flow TEM assays with EM CD4 + T cells ( B ). A , FACS plots of HDMECs stained with isotype-matched control IgG (thin lines) or anti-CD99 or -CD31 (thick lines in left panels and right panels , respectively) demonstrating knockdown of CD99 without reduction of CD31 expression. B , TEM assays. Graph on the left (VB2 − ) shows TEM of Vβ2TCR − cells (i.e., those T cells with TCR that are not activated by TSST-1) at 15 min. Graph on the right (VB2 + ) shows TEM of Vβ2TCR + cells at 60 min. Graphs display data from one representative experiment of two (VB2 − ) or three (VB2 + ) separate experiments using T cells from different donors. *** p

    Techniques Used: Transmission Electron Microscopy, Transfection, FACS, Flow Cytometry, Staining, Expressing

    27) Product Images from "The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans"

    Article Title: The Golgin GCC88 Is Required for Efficient Retrograde Transport of Cargo from the Early Endosomes to the Trans

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-06-0622

    Depletion of endogenous GCC88 by an inducible shRNA. HeLa cells (clone A8) stably expressing a tetracycline on inducible shRNA to GCC88 (tet R GCC88KD A8) were either untreated (control) or treated with 10 or 100 ng/ml doxycycline (Dox) for 96 h, and monolayers were fixed with 4% paraformaldehyde. (A) Endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 488-conjugated anti-rabbit IgG. (B) HeLa A8 cells were incubated with 10 ng/ml doxycycline for 96 h, lysed in SDS-PAGE reducing buffer, and then extracts were subjected to SDS-PAGE on a 7.5% polyacrylamide gel. Proteins were transfer to a polyvinylidene difluoride membrane and probed with rabbit anti-GCC88 antibodies using a chemiluminescence detection system. The membrane were then stripped and reprobed with anti-α-tubulin, followed by anti-GCC185 and anti-golgin-97 antibodies. Bar, 10 μm.
    Figure Legend Snippet: Depletion of endogenous GCC88 by an inducible shRNA. HeLa cells (clone A8) stably expressing a tetracycline on inducible shRNA to GCC88 (tet R GCC88KD A8) were either untreated (control) or treated with 10 or 100 ng/ml doxycycline (Dox) for 96 h, and monolayers were fixed with 4% paraformaldehyde. (A) Endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 488-conjugated anti-rabbit IgG. (B) HeLa A8 cells were incubated with 10 ng/ml doxycycline for 96 h, lysed in SDS-PAGE reducing buffer, and then extracts were subjected to SDS-PAGE on a 7.5% polyacrylamide gel. Proteins were transfer to a polyvinylidene difluoride membrane and probed with rabbit anti-GCC88 antibodies using a chemiluminescence detection system. The membrane were then stripped and reprobed with anti-α-tubulin, followed by anti-GCC185 and anti-golgin-97 antibodies. Bar, 10 μm.

    Techniques Used: shRNA, Stable Transfection, Expressing, Incubation, SDS Page

    Defect in TGN38 recycling is rescued by expression of wild-type syntaxin 6. HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and cotransfected with TGN38 and either GFP-syntaxin6 FL (GFP-Syn6 FL ) (A), GFP-syntaxin6 cyto (GFP-Syn6 cyto ) (B), or cherry-syntaxin 16 FL (Cherry-Syn16 FL ) (C) for 24 h before the internalization assay. Monolayers were incubated with monoclonal mouse anti-TGN38 antibodies for 30 min on ice, washed in PBS, and then incubated at 37°C in serum-free media 120 min to internalize the antibody–TGN38 complex. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa 568-conjugated anti-mouse IgG for 60 min. Endogenous GCC88 was stained with rabbit anti-GCC88 antibodies, followed by Alexa 647-conjugated anti-rabbit IgG. Cells with no GCC88 staining and perinuclear level of syntaxin 6 or syntaxin 16 expression were analyzed (n = 15, in duplicate). Bars, 10 μm.
    Figure Legend Snippet: Defect in TGN38 recycling is rescued by expression of wild-type syntaxin 6. HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and cotransfected with TGN38 and either GFP-syntaxin6 FL (GFP-Syn6 FL ) (A), GFP-syntaxin6 cyto (GFP-Syn6 cyto ) (B), or cherry-syntaxin 16 FL (Cherry-Syn16 FL ) (C) for 24 h before the internalization assay. Monolayers were incubated with monoclonal mouse anti-TGN38 antibodies for 30 min on ice, washed in PBS, and then incubated at 37°C in serum-free media 120 min to internalize the antibody–TGN38 complex. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa 568-conjugated anti-mouse IgG for 60 min. Endogenous GCC88 was stained with rabbit anti-GCC88 antibodies, followed by Alexa 647-conjugated anti-rabbit IgG. Cells with no GCC88 staining and perinuclear level of syntaxin 6 or syntaxin 16 expression were analyzed (n = 15, in duplicate). Bars, 10 μm.

    Techniques Used: Expressing, Incubation, Staining

    Shiga toxin is transported efficiently to the Golgi in GCC88-depleted cells. HeLa A8 cells were either untreated (control) (A) or incubated with 10 ng/ml doxycycline for 96 h (GCC88 depleted) (B). Monolayers were incubated with Cy3-conjugated STx-B for 45 min on ice, and then they were either fixed immediately (0 min) or incubated at 37°C for either 20 or 60 min, followed by fixation in 4% paraformaldehyde. Cells were stained with monoclonal antibodies to GM130 followed by Alexa-conjugated anti-mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Shiga toxin is transported efficiently to the Golgi in GCC88-depleted cells. HeLa A8 cells were either untreated (control) (A) or incubated with 10 ng/ml doxycycline for 96 h (GCC88 depleted) (B). Monolayers were incubated with Cy3-conjugated STx-B for 45 min on ice, and then they were either fixed immediately (0 min) or incubated at 37°C for either 20 or 60 min, followed by fixation in 4% paraformaldehyde. Cells were stained with monoclonal antibodies to GM130 followed by Alexa-conjugated anti-mouse IgG. Bars, 10 μm.

    Techniques Used: Incubation, Staining

    Anterograde transport of E-cadherin in GCC88-depleted cells is unaffected. HeLa A8 cells were either untreated (control) or incubated with 10 ng/ml Dox for 72 h and then transfected with Ecad-GFP for 24 h before staining. (A) Monolayers were fixed with 4% paraformaldehyde, permeabilized, and endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 568-conjugated anti-rabbit IgG and Ecad-GFP by GFP fluorescence. (B) Monolayers were fixed and stained with monoclonal anti-Ecad antibodies followed by Alexa 568-conjugated goat ant-mouse IgG. Fixed monolayers were then permeabilized and stained with rabbit anti-GCC88 antibodies followed by Alexa 647-conjugated goat anti-rabbit IgG. Bars, 10 μm.
    Figure Legend Snippet: Anterograde transport of E-cadherin in GCC88-depleted cells is unaffected. HeLa A8 cells were either untreated (control) or incubated with 10 ng/ml Dox for 72 h and then transfected with Ecad-GFP for 24 h before staining. (A) Monolayers were fixed with 4% paraformaldehyde, permeabilized, and endogenous GCC88 was detected with rabbit anti-GCC88 antibodies followed by Alexa 568-conjugated anti-rabbit IgG and Ecad-GFP by GFP fluorescence. (B) Monolayers were fixed and stained with monoclonal anti-Ecad antibodies followed by Alexa 568-conjugated goat ant-mouse IgG. Fixed monolayers were then permeabilized and stained with rabbit anti-GCC88 antibodies followed by Alexa 647-conjugated goat anti-rabbit IgG. Bars, 10 μm.

    Techniques Used: Incubation, Transfection, Staining, Fluorescence

    Syntaxin 6 depletion impairs TGN38, but not Shiga toxin, trafficking to the Golgi apparatus. (A) HeLa cells were transfected with syntaxin 6 siRNA for 72 h, fixed in 4% paraformaldehyde, saponin permeablized, and stained with monoclonal anti-syntaxin 6 antibodies followed by Alexa-conjugated mouse IgG. (B) HeLa cells transfected with siRNA as described above for 48 h, and then they were transfected a second time with CFP-TGN38 for a further 24 h. Monolayers were then incubated with monoclonal mouse anti-TGN38 antibodies on ice for 30 min, washed in PBS, and incubated in serum-free media at 37°C for 120 min. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa-conjugated anti-mouse IgG. Endogenous GMAP-210 was stained with rabbit anti-GMAP-210, followed by Alexa-conjugated anti-rabbit IgG. C) HeLa cells were transfected with syntaxin 6 siRNA for 72 h and incubated with Cy3-conjugated STx-B for 45 min on ice and then either fixed immediately (0 min) or incubated at 37°C for 60 min followed by fixation. Cells were stained with monoclonal antibodies to GM130 followed by Alexa 647-conjugated mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Syntaxin 6 depletion impairs TGN38, but not Shiga toxin, trafficking to the Golgi apparatus. (A) HeLa cells were transfected with syntaxin 6 siRNA for 72 h, fixed in 4% paraformaldehyde, saponin permeablized, and stained with monoclonal anti-syntaxin 6 antibodies followed by Alexa-conjugated mouse IgG. (B) HeLa cells transfected with siRNA as described above for 48 h, and then they were transfected a second time with CFP-TGN38 for a further 24 h. Monolayers were then incubated with monoclonal mouse anti-TGN38 antibodies on ice for 30 min, washed in PBS, and incubated in serum-free media at 37°C for 120 min. Monolayers were fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa-conjugated anti-mouse IgG. Endogenous GMAP-210 was stained with rabbit anti-GMAP-210, followed by Alexa-conjugated anti-rabbit IgG. C) HeLa cells were transfected with syntaxin 6 siRNA for 72 h and incubated with Cy3-conjugated STx-B for 45 min on ice and then either fixed immediately (0 min) or incubated at 37°C for 60 min followed by fixation. Cells were stained with monoclonal antibodies to GM130 followed by Alexa 647-conjugated mouse IgG. Bars, 10 μm.

    Techniques Used: Transfection, Staining, Incubation

    Depletion of GCC88 results in mislocalization of syntaxin 6. (A–E) HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and then fixed in 4% paraformaldehyde and permeablized. Fixed monolayers were stained for endogenous VAMP4 (A), VAMP3 (B), Vti1a (C), syntaxin 6 (D), and syntaxin 16 (E), with rabbit polyclonal antibodies to VAMP4, VAMP3, and syntaxin 16, respectively, followed by Alexa 568-conjugated rabbit IgG and mouse monoclonal antibodies to vti1a and syntaxin 6, respectively, followed by Alexa 568-conjugated anti-mouse IgG. (F) Untransfected HeLa cells (control) or HeLa cells transfected with p230 siRNA (p230 depleted) for 72 h were fixed in 4% paraformaldehyde and saponin permeabilized. Endogenous p230 was stained with human anti-p230 antibodies followed by FITC-conjugated anti-human IgG and syntaxin 6 stained with monoclonal mouse anti-syntaxin 6, followed by Alexa 568-conjugated anti-mouse IgG. Bars, 10 μm.
    Figure Legend Snippet: Depletion of GCC88 results in mislocalization of syntaxin 6. (A–E) HeLa A8 were either untreated (control) or incubated in 10 ng/ml doxycycline for 96 h (GCC88 depleted) and then fixed in 4% paraformaldehyde and permeablized. Fixed monolayers were stained for endogenous VAMP4 (A), VAMP3 (B), Vti1a (C), syntaxin 6 (D), and syntaxin 16 (E), with rabbit polyclonal antibodies to VAMP4, VAMP3, and syntaxin 16, respectively, followed by Alexa 568-conjugated rabbit IgG and mouse monoclonal antibodies to vti1a and syntaxin 6, respectively, followed by Alexa 568-conjugated anti-mouse IgG. (F) Untransfected HeLa cells (control) or HeLa cells transfected with p230 siRNA (p230 depleted) for 72 h were fixed in 4% paraformaldehyde and saponin permeabilized. Endogenous p230 was stained with human anti-p230 antibodies followed by FITC-conjugated anti-human IgG and syntaxin 6 stained with monoclonal mouse anti-syntaxin 6, followed by Alexa 568-conjugated anti-mouse IgG. Bars, 10 μm.

    Techniques Used: Incubation, Staining, Transfection

    28) Product Images from "Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells"

    Article Title: Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20144322

    Confirmation of the allografted quasi-natural cell block in the recipient body. To confirm allogenicity of the transplanted quasi-natural cell block, gender-specific probes were used to distinguish the male-derived allografted tissue from the recipient female mouse by FISH along with DAPI staining of the nucleus ( A, B ). Green spots indicate male Y chromosome probe in the corresponding female tissue. For intercellular communication capability in the allografted tissue, specimens were stained with specific probes for signaling marker connexin43, in the quasi-natural tissue ( C ) along with native heart tissue ( D ). Immunostaining of the allografted quasi-natural tissue with epithelial linage marker CD31 revealed vascular network assembly as indicated ( E ). Immunostaining of the allografted quasi-natural tissue further showed that most of cells were stained with anti-vimentin, mesodermal lineage marker, but not with anti-cytokeratin ( F ). Images for individual channels (connexin43 with alexa 488 is green, cd31 with alexa 568 is red, vimentin with alexa 488 is green, cytokeratin with alexa 568 is red) are shown on the left, and main panels show the merged image containing all channels plus DIC. The cells nuclei were visualized with DAPI (blue). Scale bar: ( A, C, D, E and F ) 20 μm; ( B ) 100 μm.
    Figure Legend Snippet: Confirmation of the allografted quasi-natural cell block in the recipient body. To confirm allogenicity of the transplanted quasi-natural cell block, gender-specific probes were used to distinguish the male-derived allografted tissue from the recipient female mouse by FISH along with DAPI staining of the nucleus ( A, B ). Green spots indicate male Y chromosome probe in the corresponding female tissue. For intercellular communication capability in the allografted tissue, specimens were stained with specific probes for signaling marker connexin43, in the quasi-natural tissue ( C ) along with native heart tissue ( D ). Immunostaining of the allografted quasi-natural tissue with epithelial linage marker CD31 revealed vascular network assembly as indicated ( E ). Immunostaining of the allografted quasi-natural tissue further showed that most of cells were stained with anti-vimentin, mesodermal lineage marker, but not with anti-cytokeratin ( F ). Images for individual channels (connexin43 with alexa 488 is green, cd31 with alexa 568 is red, vimentin with alexa 488 is green, cytokeratin with alexa 568 is red) are shown on the left, and main panels show the merged image containing all channels plus DIC. The cells nuclei were visualized with DAPI (blue). Scale bar: ( A, C, D, E and F ) 20 μm; ( B ) 100 μm.

    Techniques Used: Blocking Assay, Derivative Assay, Fluorescence In Situ Hybridization, Staining, Marker, Immunostaining

    29) Product Images from "Combinatorial interactions of genetic variants in human cardiomyopathy"

    Article Title: Combinatorial interactions of genetic variants in human cardiomyopathy

    Journal: Nature biomedical engineering

    doi: 10.1038/s41551-019-0348-9

    hPSC-derived cardiomyocytes harbouring TEK ; VCL genetic variants exhibit functional and sarcomeric organization defects. a–f , Single-cardiomyocyte traction force microscopy studies show that TV-Dhet and VCL VFS1 /+ hPSC-derived cardiomyocytes exhibit reduced contractility as detected by tangential stress heat maps ( a,d ), strain energy time courses ( b,e ) and peak strain energy analyses ( c,f ). g–j , Alpha-actinin immunostaining (green; g,i ) and quantitation of the sarcomeric organization ( h,j ) reveal that sarcomeres of TV-Dhet and VCL VFS1 /+ hPSC-derived cardiomyocytes are more disorganized than that of WT and CTRL cardiomyocytes. Control hESC-derived ( WT ) and CRISPR-edited-hESC-derived cardiomyocytes are represented in a–c,g,h . Control hiPSC-derived (CTRL) and cardiomyopathy-affected patient ( TV-Dhet ) hiPSC-derived cardiomyocytes are represented in d–f,i,j . Data are presented as interquartile range (box) around the mean (horizontal line), whiskers represent the minimum and maximum of the dataset, with the individual data points superimposed. * P
    Figure Legend Snippet: hPSC-derived cardiomyocytes harbouring TEK ; VCL genetic variants exhibit functional and sarcomeric organization defects. a–f , Single-cardiomyocyte traction force microscopy studies show that TV-Dhet and VCL VFS1 /+ hPSC-derived cardiomyocytes exhibit reduced contractility as detected by tangential stress heat maps ( a,d ), strain energy time courses ( b,e ) and peak strain energy analyses ( c,f ). g–j , Alpha-actinin immunostaining (green; g,i ) and quantitation of the sarcomeric organization ( h,j ) reveal that sarcomeres of TV-Dhet and VCL VFS1 /+ hPSC-derived cardiomyocytes are more disorganized than that of WT and CTRL cardiomyocytes. Control hESC-derived ( WT ) and CRISPR-edited-hESC-derived cardiomyocytes are represented in a–c,g,h . Control hiPSC-derived (CTRL) and cardiomyopathy-affected patient ( TV-Dhet ) hiPSC-derived cardiomyocytes are represented in d–f,i,j . Data are presented as interquartile range (box) around the mean (horizontal line), whiskers represent the minimum and maximum of the dataset, with the individual data points superimposed. * P

    Techniques Used: Derivative Assay, Functional Assay, Microscopy, Immunostaining, Quantitation Assay, CRISPR

    30) Product Images from "Telocytes constitute a widespread interstitial meshwork in the lamina propria and underlying striated muscle of human tongue"

    Article Title: Telocytes constitute a widespread interstitial meshwork in the lamina propria and underlying striated muscle of human tongue

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-42415-3

    Double immunofluorescence staining of human tongue tissue sections. ( A–C ) CD34 (green) and CD31 (red) immunostaining with 4′,6-diamidino-2-phenylindole (DAPI; blue) counterstain for nuclei. Telocytes (TCs)/CD34+ stromal cells in the tongue lamina propria ( A,B ) and skeletal muscle ( C ) lack CD31 immunoreactivity. The endothelial cells of blood vessels (arrows) are CD34+/CD31+ ( A,C ). At variance with double stained blood vessels (BV), the endothelium of lymphatic vessels (LV) is CD34−/CD31+ ( B ). Some leukocytes are also CD31+ ( A,B ). ( D–F ) CD34 (green) and α-smooth muscle actin (α-SMA; red) immunofluorescence labeling with DAPI counterstain. CD34+ TCs do not coexpress α-SMA. ( D ) CD34+ TCs envelop secretory salivary gland units outside of α-SMA + myoepithelial cells. ( E,F ) CD34+ TCs externally encircle the vascular smooth muscle cell layer of arterioles (arrows; higher magnification in the inset). ( G–I ) CD34 (green) and c-kit/CD117 (red) immunofluorescence with DAPI counterstain. The CD34+ TC meshwork is immunophenotypically negative for the c-kit/CD117 marker. c-kit/CD117 is detectable only in oval/round-shaped mast cells (arrows) often in close relationship with CD34+ TC processes (higher magnification in the inset). Scale bar: 50 µm ( A–I ).
    Figure Legend Snippet: Double immunofluorescence staining of human tongue tissue sections. ( A–C ) CD34 (green) and CD31 (red) immunostaining with 4′,6-diamidino-2-phenylindole (DAPI; blue) counterstain for nuclei. Telocytes (TCs)/CD34+ stromal cells in the tongue lamina propria ( A,B ) and skeletal muscle ( C ) lack CD31 immunoreactivity. The endothelial cells of blood vessels (arrows) are CD34+/CD31+ ( A,C ). At variance with double stained blood vessels (BV), the endothelium of lymphatic vessels (LV) is CD34−/CD31+ ( B ). Some leukocytes are also CD31+ ( A,B ). ( D–F ) CD34 (green) and α-smooth muscle actin (α-SMA; red) immunofluorescence labeling with DAPI counterstain. CD34+ TCs do not coexpress α-SMA. ( D ) CD34+ TCs envelop secretory salivary gland units outside of α-SMA + myoepithelial cells. ( E,F ) CD34+ TCs externally encircle the vascular smooth muscle cell layer of arterioles (arrows; higher magnification in the inset). ( G–I ) CD34 (green) and c-kit/CD117 (red) immunofluorescence with DAPI counterstain. The CD34+ TC meshwork is immunophenotypically negative for the c-kit/CD117 marker. c-kit/CD117 is detectable only in oval/round-shaped mast cells (arrows) often in close relationship with CD34+ TC processes (higher magnification in the inset). Scale bar: 50 µm ( A–I ).

    Techniques Used: Double Immunofluorescence Staining, Immunostaining, Staining, Immunofluorescence, Labeling, Marker

    Double immunofluorescence staining of human tongue tissue sections. ( A–F ) CD34 (green) and platelet-derived growth factor receptor α (PDGFRα; red) immunofluorescence with 4′,6-diamidino-2-phenylindole (DAPI; blue) counterstain for nuclei. Single green and red images are shown in ( A,D , B,E ), respectively, while overlay images are shown in ( C,F ). All telocytes (TCs)/CD34+ stromal cells within the tongue stromal compartment are PDGFRα+. Colocalization of CD34 and PDGFRα on the TC surface gives rise to yellow staining. Scale bar: 50 µm ( A–F ).
    Figure Legend Snippet: Double immunofluorescence staining of human tongue tissue sections. ( A–F ) CD34 (green) and platelet-derived growth factor receptor α (PDGFRα; red) immunofluorescence with 4′,6-diamidino-2-phenylindole (DAPI; blue) counterstain for nuclei. Single green and red images are shown in ( A,D , B,E ), respectively, while overlay images are shown in ( C,F ). All telocytes (TCs)/CD34+ stromal cells within the tongue stromal compartment are PDGFRα+. Colocalization of CD34 and PDGFRα on the TC surface gives rise to yellow staining. Scale bar: 50 µm ( A–F ).

    Techniques Used: Double Immunofluorescence Staining, Derivative Assay, Immunofluorescence, Staining

    Immunohistochemical localization of telocytes (TCs)/CD34+ stromal cells in the human tongue lamina propria. ( A ) Hematoxylin and eosin staining testifying the normal appearance of the lamina propria connective tissue. ( B – H ) CD34 immunohistochemistry with hematoxylin counterstain. ( B ) An extensive CD34+ cell meshwork is finely distributed throughout the tongue lamina propria. Inset: negative control. ( C–E ) CD34+ interstitial cells exhibit the typical TC morphology ( i.e . spindle-shaped cells with a small nucleated body and very long and thin moniliform/varicose telopodes). ( C ) Note the almost continuous TC layer along the basement membrane beneath the oral mucosa epithelium (dashed arrows). ( D ) TCs are in close relationship with histiocytes/mononuclear cells. ( E ) CD34+ TCs are particularly concentrated around blood vessels (BV) and lymphatic vessels (LV) (inset). CD34 immunoreactivity is detected also in blood vascular endothelium, but not in lymphatic endothelium. ( F ) TCs are arranged to delimit externally the mucosa-associated lymphoid tissue (MALT) aggregates (arrows; higher magnification in the inset). ( G ) CD34+ TCs closely surround the secretory units of mucous glands (MG) and serous glands (SG) (inset). ( H ) TCs are also distributed around salivary gland excretory ducts. Scale bar: 200 µm ( A ), 100 µm ( B,F ), 50 µm ( C–E,G,H ).
    Figure Legend Snippet: Immunohistochemical localization of telocytes (TCs)/CD34+ stromal cells in the human tongue lamina propria. ( A ) Hematoxylin and eosin staining testifying the normal appearance of the lamina propria connective tissue. ( B – H ) CD34 immunohistochemistry with hematoxylin counterstain. ( B ) An extensive CD34+ cell meshwork is finely distributed throughout the tongue lamina propria. Inset: negative control. ( C–E ) CD34+ interstitial cells exhibit the typical TC morphology ( i.e . spindle-shaped cells with a small nucleated body and very long and thin moniliform/varicose telopodes). ( C ) Note the almost continuous TC layer along the basement membrane beneath the oral mucosa epithelium (dashed arrows). ( D ) TCs are in close relationship with histiocytes/mononuclear cells. ( E ) CD34+ TCs are particularly concentrated around blood vessels (BV) and lymphatic vessels (LV) (inset). CD34 immunoreactivity is detected also in blood vascular endothelium, but not in lymphatic endothelium. ( F ) TCs are arranged to delimit externally the mucosa-associated lymphoid tissue (MALT) aggregates (arrows; higher magnification in the inset). ( G ) CD34+ TCs closely surround the secretory units of mucous glands (MG) and serous glands (SG) (inset). ( H ) TCs are also distributed around salivary gland excretory ducts. Scale bar: 200 µm ( A ), 100 µm ( B,F ), 50 µm ( C–E,G,H ).

    Techniques Used: Immunohistochemistry, Staining, Negative Control

    Immunohistochemical localization of telocytes (TCs)/CD34+ stromal cells in the interstitium of the human tongue striated muscle. ( A ) Hematoxylin and eosin staining demonstrating the normal appearance of the tongue muscle. ( B–H ) CD34 immunohistochemistry with hematoxylin counterstain. ( B,C ) A diffuse CD34+ reticular network is evident in the perimysium encasing skeletal muscle bundles ( B ) and around intramuscular vessels (dashed arrows) and nerves (arrows) ( C ). Inset: negative control. ( D–F ) The endomysium is populated by a dense meshwork of CD34+ TCs projecting long and moniliform telopodes in close relationship with skeletal muscle fibers. ( G ) CD34+ TCs intimately encircle intramuscular arterioles (BV, blood vessels). ( H ) TCs form an outer sheath (arrows) for intramuscular nerves (NE) and ganglia (asterisk). Scale bar: 200 µm ( A ), 100 µm ( B,C ), 50 µm ( D–H ).
    Figure Legend Snippet: Immunohistochemical localization of telocytes (TCs)/CD34+ stromal cells in the interstitium of the human tongue striated muscle. ( A ) Hematoxylin and eosin staining demonstrating the normal appearance of the tongue muscle. ( B–H ) CD34 immunohistochemistry with hematoxylin counterstain. ( B,C ) A diffuse CD34+ reticular network is evident in the perimysium encasing skeletal muscle bundles ( B ) and around intramuscular vessels (dashed arrows) and nerves (arrows) ( C ). Inset: negative control. ( D–F ) The endomysium is populated by a dense meshwork of CD34+ TCs projecting long and moniliform telopodes in close relationship with skeletal muscle fibers. ( G ) CD34+ TCs intimately encircle intramuscular arterioles (BV, blood vessels). ( H ) TCs form an outer sheath (arrows) for intramuscular nerves (NE) and ganglia (asterisk). Scale bar: 200 µm ( A ), 100 µm ( B,C ), 50 µm ( D–H ).

    Techniques Used: Immunohistochemistry, Staining, Negative Control

    31) Product Images from "Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis"

    Article Title: Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis

    Journal: Cell reports

    doi: 10.1016/j.celrep.2016.12.051

    CQ Induced Par-4 Secretion Is Dependent on Rab8b (A) CQ induced Par-4 secretion by a Rab8b-dependent mechanism. Rab8 wild-type (WT), Rab8b −/− , or Rab8a −/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr, and the CM or lysates were examined by western blot analysis with the indicated antibodies. (B) Induction of Par-4 secretion in response to CQ in Rab8b null cells was restored by re-introduction of Rab8b. Rab8b null MEFs were transiently transfected with GFP-mouse Rab8b (GFP-mRab8b) expression construct or GFP-expression construct for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies. (C) Par-4 secretion in response to CQ was inhibited by Rab8b siRNAs. Wild-type MEFs were transfected with siRNA duplexes from two different sources, Dharmacon (D) and Santa Cruz Biotechnology (SC), or with scrambled siRNA duplexes for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies. (D) Introduction of human Rab8b in Rab8b-knockdown MEFs resulted in restoration of Par-4 secretion in response to CQ. MEFs were transfected with the indicated siRNAs, 24 hr later, they were re-transfected with GFP-human Rab8b (GFP-hRab8b) or GFP expression construct, and the transfectants were treated for 24 hr with CQ (25 µM) or vehicle. Western blot analysis of the CM and lysates was performed by using the indicated antibodies. Knockdown of endogenous Rab8b was confirmed with the Rab8b antibody, and expression of GFP-hRab8b was detected with the GFP antibody. See also Figure S5 .
    Figure Legend Snippet: CQ Induced Par-4 Secretion Is Dependent on Rab8b (A) CQ induced Par-4 secretion by a Rab8b-dependent mechanism. Rab8 wild-type (WT), Rab8b −/− , or Rab8a −/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr, and the CM or lysates were examined by western blot analysis with the indicated antibodies. (B) Induction of Par-4 secretion in response to CQ in Rab8b null cells was restored by re-introduction of Rab8b. Rab8b null MEFs were transiently transfected with GFP-mouse Rab8b (GFP-mRab8b) expression construct or GFP-expression construct for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies. (C) Par-4 secretion in response to CQ was inhibited by Rab8b siRNAs. Wild-type MEFs were transfected with siRNA duplexes from two different sources, Dharmacon (D) and Santa Cruz Biotechnology (SC), or with scrambled siRNA duplexes for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies. (D) Introduction of human Rab8b in Rab8b-knockdown MEFs resulted in restoration of Par-4 secretion in response to CQ. MEFs were transfected with the indicated siRNAs, 24 hr later, they were re-transfected with GFP-human Rab8b (GFP-hRab8b) or GFP expression construct, and the transfectants were treated for 24 hr with CQ (25 µM) or vehicle. Western blot analysis of the CM and lysates was performed by using the indicated antibodies. Knockdown of endogenous Rab8b was confirmed with the Rab8b antibody, and expression of GFP-hRab8b was detected with the GFP antibody. See also Figure S5 .

    Techniques Used: Western Blot, Transfection, Expressing, Construct

    Rab8b Is a Direct Downstream Target of p53, which Is Activated by CQ (A) CQ induced Rab8b protein and mRNA levels in a p53-dependent manner. Wild-type (p53 +/+ ) or p53 −/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr; and either the lysates were examined by western blot analysis with the indicated antibodies (left panel), or mRNA prepared from the cells was examined by real-time qRT-PCR (right panel). *p
    Figure Legend Snippet: Rab8b Is a Direct Downstream Target of p53, which Is Activated by CQ (A) CQ induced Rab8b protein and mRNA levels in a p53-dependent manner. Wild-type (p53 +/+ ) or p53 −/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr; and either the lysates were examined by western blot analysis with the indicated antibodies (left panel), or mRNA prepared from the cells was examined by real-time qRT-PCR (right panel). *p

    Techniques Used: Western Blot, Quantitative RT-PCR

    32) Product Images from "Rhesus Rhadinovirus Infection of Rhesus Fibroblasts Occurs through Clathrin-Mediated Endocytosis ▿"

    Article Title: Rhesus Rhadinovirus Infection of Rhesus Fibroblasts Occurs through Clathrin-Mediated Endocytosis ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01429-10

    RRV particles are colocalized with markers of clathrin-mediated endocytosis during entry into RFs. (A to D) Detection of colocalization of RRV-RFP particles with Alexa Fluor 488-transferrin (A), clathrin (B), and EEA1 (C), but not with Alexa Fluor 488-CTB
    Figure Legend Snippet: RRV particles are colocalized with markers of clathrin-mediated endocytosis during entry into RFs. (A to D) Detection of colocalization of RRV-RFP particles with Alexa Fluor 488-transferrin (A), clathrin (B), and EEA1 (C), but not with Alexa Fluor 488-CTB

    Techniques Used: CtB Assay

    33) Product Images from "Effects of Cadmium on ZO-1 Tight Junction Integrity of the Blood Brain Barrier"

    Article Title: Effects of Cadmium on ZO-1 Tight Junction Integrity of the Blood Brain Barrier

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20236010

    Effect of CdCl 2 on ZO-1, F-actin, and vimentin localization in RBE4 cells. On the left: changes in the distribution of ZO-1 (red) ( A ), F-actin (green) ( B ), and vimentin (red) ( C ) in RBE4 cells treated with 10 μM CdCl 2 for 8 and 16 h. Asterisks show holes formed between endothelial cells. In ( A ), arrows point to morphological alterations in intercellular junctions, indicative for loss of junctional function. In ( B ), note the Cd-dependent appearance of numerous stress fibers. In ( C ), Cd-dependent polarization of vimentin with the formation of clumps and aggregates (arrowheads) at the edges of the cell. Nuclei were stained with DAPI (blue). Total magnification 400×; n = 135; scale bar: 50 μm. On the right: representative western blot of the effects of CdCl 2 on ZO-1, F-actin, and vimentin protein levels after 8 and 16 h of treatment. Bars represent the mean ± S.E.M., n = 4. Control condition was arbitrarily set at 100%.
    Figure Legend Snippet: Effect of CdCl 2 on ZO-1, F-actin, and vimentin localization in RBE4 cells. On the left: changes in the distribution of ZO-1 (red) ( A ), F-actin (green) ( B ), and vimentin (red) ( C ) in RBE4 cells treated with 10 μM CdCl 2 for 8 and 16 h. Asterisks show holes formed between endothelial cells. In ( A ), arrows point to morphological alterations in intercellular junctions, indicative for loss of junctional function. In ( B ), note the Cd-dependent appearance of numerous stress fibers. In ( C ), Cd-dependent polarization of vimentin with the formation of clumps and aggregates (arrowheads) at the edges of the cell. Nuclei were stained with DAPI (blue). Total magnification 400×; n = 135; scale bar: 50 μm. On the right: representative western blot of the effects of CdCl 2 on ZO-1, F-actin, and vimentin protein levels after 8 and 16 h of treatment. Bars represent the mean ± S.E.M., n = 4. Control condition was arbitrarily set at 100%.

    Techniques Used: Staining, Western Blot

    34) Product Images from "Nuclear localization of Annexin A7 during murine brain development"

    Article Title: Nuclear localization of Annexin A7 during murine brain development

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-6-25

    Annexin A7 immunostaining in the cerebellum of adult mice. ( A ) Low magnification of the cerebellum presents an Annexin A7 expression mainly in cells of the stratum granulosum and laminae medullares. ( B ) Corresponding section stained for GFAP. ( C ) Higher magnification of folia of the cerebellum, where a polyclonal anti-AnnexinA7 antibody was used; square, a higher magnification of acorresponding area stained with mAb 203–217 is given in (D). Between stratum granulosum and stratum moleculare the layer of Purkinje-cells (stratum neuronorum piriformium (ganglionare)) can be observed. ( D ) Staining of the band of Purkinje-cells (arrows). The positive Annexin A7-stain in the stratum granulosum is due to staining of the nuclei of neurons. ( E ) In addition to an intense staining of the nucleus, the cell body is AnnexinA7-positive including both dendrites of the Purkinje-cell shown. ( F ) Corresponding section from an AnxA7 -/- mouse. ( G ) AnnexinA7 staining of axons (arrowheads) running from the laminae medullares to the Purkinje-cell layer located in the round end of a convolution. Sections A, D, E, F, G were stained with mAb 203–217.
    Figure Legend Snippet: Annexin A7 immunostaining in the cerebellum of adult mice. ( A ) Low magnification of the cerebellum presents an Annexin A7 expression mainly in cells of the stratum granulosum and laminae medullares. ( B ) Corresponding section stained for GFAP. ( C ) Higher magnification of folia of the cerebellum, where a polyclonal anti-AnnexinA7 antibody was used; square, a higher magnification of acorresponding area stained with mAb 203–217 is given in (D). Between stratum granulosum and stratum moleculare the layer of Purkinje-cells (stratum neuronorum piriformium (ganglionare)) can be observed. ( D ) Staining of the band of Purkinje-cells (arrows). The positive Annexin A7-stain in the stratum granulosum is due to staining of the nuclei of neurons. ( E ) In addition to an intense staining of the nucleus, the cell body is AnnexinA7-positive including both dendrites of the Purkinje-cell shown. ( F ) Corresponding section from an AnxA7 -/- mouse. ( G ) AnnexinA7 staining of axons (arrowheads) running from the laminae medullares to the Purkinje-cell layer located in the round end of a convolution. Sections A, D, E, F, G were stained with mAb 203–217.

    Techniques Used: Immunostaining, Mouse Assay, Expressing, Staining

    Subcellular localization of Annexin A7 in embryos E13, E15 and E16. Paraffin sections of embryonic brain were stained with purified mAb 203–217. Annexin A7 was visualized with Alexa Fluor 488-conjugated secondary antibody. ( A ) Overview, at E16 the immature GFAP-negative cells of the forming cerebral neocortex (b) surrounding the lateral ventricle (a) are strongly stained; square, a higher magnification of this area is given in (I). ( B ) Higher magnification of the earlier stage E13 (H) shows, that the cells are stained in the cytosol (arrowhead). ( C ) Two days later at E15 the cells have rounded up, and Annexin A7 stays in the cytosol. ( D ) At E16 the first nuclear staining becomes apparent (arrowhead) in cells of the intermediate zone located between ventricular germinative zone and marginal neopallial cortex as seen in (I), oval. ( E-G ) Confocal microscopy confirms the results in B-D. ( H ) Overview, at E13 the immature GFAP-negative cells are strongly stained; a, lateral ventricle. ( I ) Higher magnification of a cortical section of (A, square) demonstrating ventricular, intermediate, and marginal zones; oval, a higher magnification of this area is given in (D,G).
    Figure Legend Snippet: Subcellular localization of Annexin A7 in embryos E13, E15 and E16. Paraffin sections of embryonic brain were stained with purified mAb 203–217. Annexin A7 was visualized with Alexa Fluor 488-conjugated secondary antibody. ( A ) Overview, at E16 the immature GFAP-negative cells of the forming cerebral neocortex (b) surrounding the lateral ventricle (a) are strongly stained; square, a higher magnification of this area is given in (I). ( B ) Higher magnification of the earlier stage E13 (H) shows, that the cells are stained in the cytosol (arrowhead). ( C ) Two days later at E15 the cells have rounded up, and Annexin A7 stays in the cytosol. ( D ) At E16 the first nuclear staining becomes apparent (arrowhead) in cells of the intermediate zone located between ventricular germinative zone and marginal neopallial cortex as seen in (I), oval. ( E-G ) Confocal microscopy confirms the results in B-D. ( H ) Overview, at E13 the immature GFAP-negative cells are strongly stained; a, lateral ventricle. ( I ) Higher magnification of a cortical section of (A, square) demonstrating ventricular, intermediate, and marginal zones; oval, a higher magnification of this area is given in (D,G).

    Techniques Used: Staining, Purification, Confocal Microscopy

    Annexin A7 is present in neurons and astrocytes of the cortex temporalis and hippocampal formation of 10-weeks-old mice. ( A ) Low magnification of the cortex temporalis presents an Annexin A7 expression in cells of the pial border, in neurons of all six isocortical laminae, and a weak signal in the adjacent white matter. ( B ) Corresponding section stained with GFAP. ( C ) Staining in the Stratum pyramidalis (a) and in the dentate gyrus of the hippocampus; square, a higher magnification of acorresponding area is given in (G,H). An intense Annexin A7 immunostaining is detectable. ( D ) Corresponding section stained with secondary antibody only. ( E ) Presence of Annexin A7 in pyramidal neurons (lamina pyramidalis externa) of the isocortex temporalis. These neurons were identified based on their morphology, distribution and lack of GFAP staining. AnnexinA7 exhibits a punctate staining, which is pronounced in the nucleus (arrowhead). ( F ) Higher magnification of image (A) also shows Annexin A7 in nuclei of neurons (lamina granularis externa (corpuscularis), arrowhead) and in the cytoplasm and nuclei of astrocytes (lamina molecularis, arrow; GFAP-confirmed). ( G ) Higher magnification of the pyramidal neurons in the hippocampus confirms the presence of the Annexin A7 protein in the nucleus (arrowhead) of mature neurons. ( H ) To further confirm this, a similar section derived from an AnxA7 -/- mouse was stained with the annexin specific antibody and lacked the nuclear signal. The residual stain of the tissue is unspecific, as it is also observed in controls of the AnxA7 -/- brain omitting the primary antibody (data not shown). All paraffin sections were stained with mAb 203–217 (A, C, E, F, G) or anti-GFAP-antibody (B). The hippocampal control section (D) lacks the primary antibody.
    Figure Legend Snippet: Annexin A7 is present in neurons and astrocytes of the cortex temporalis and hippocampal formation of 10-weeks-old mice. ( A ) Low magnification of the cortex temporalis presents an Annexin A7 expression in cells of the pial border, in neurons of all six isocortical laminae, and a weak signal in the adjacent white matter. ( B ) Corresponding section stained with GFAP. ( C ) Staining in the Stratum pyramidalis (a) and in the dentate gyrus of the hippocampus; square, a higher magnification of acorresponding area is given in (G,H). An intense Annexin A7 immunostaining is detectable. ( D ) Corresponding section stained with secondary antibody only. ( E ) Presence of Annexin A7 in pyramidal neurons (lamina pyramidalis externa) of the isocortex temporalis. These neurons were identified based on their morphology, distribution and lack of GFAP staining. AnnexinA7 exhibits a punctate staining, which is pronounced in the nucleus (arrowhead). ( F ) Higher magnification of image (A) also shows Annexin A7 in nuclei of neurons (lamina granularis externa (corpuscularis), arrowhead) and in the cytoplasm and nuclei of astrocytes (lamina molecularis, arrow; GFAP-confirmed). ( G ) Higher magnification of the pyramidal neurons in the hippocampus confirms the presence of the Annexin A7 protein in the nucleus (arrowhead) of mature neurons. ( H ) To further confirm this, a similar section derived from an AnxA7 -/- mouse was stained with the annexin specific antibody and lacked the nuclear signal. The residual stain of the tissue is unspecific, as it is also observed in controls of the AnxA7 -/- brain omitting the primary antibody (data not shown). All paraffin sections were stained with mAb 203–217 (A, C, E, F, G) or anti-GFAP-antibody (B). The hippocampal control section (D) lacks the primary antibody.

    Techniques Used: Mouse Assay, Expressing, Staining, Immunostaining, Derivative Assay

    35) Product Images from "Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence"

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00864-13

    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Figure Legend Snippet: Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Techniques Used: Confocal Microscopy, Labeling, Incubation, Infection, Invasion Assay

    Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Figure Legend Snippet: Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Techniques Used: Confocal Microscopy, Clonogenic Cell Survival Assay, Labeling, Infection

    36) Product Images from "Toll-Like Receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors"

    Article Title: Toll-Like Receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors

    Journal: Cancer science

    doi: 10.1111/j.1349-7006.2010.01491.x

    Colocalization analysis in Hs578T human breast cancer cells. (a) Unstimulated cells expressing eCFP tagged TLR9 were stained with Endoplasmatic Reticulum Tracker dye (blue, blue-white tracker). Untransfected cells were stimulated with (b) CpG-DNA (c) GpC-DNA or (d) mCpG-DNA. Cells were stained with (b-d) anti-TLR9 (red, Alexa 568) and (b, c) DAPI nucleic-acid stain. (e) Stimulated, untransfected cells were stained with anti-TLR9 (red, Alexa 568), DAPI nucleic-acid stain and anti-EEA1 an early endosome marker (green, FITC). (f) Unstimulated, untransfected cells were stained with anti-TLR9 (red, Alexa 568) and anti-EEA1 an early endosome marker (green, FITC).
    Figure Legend Snippet: Colocalization analysis in Hs578T human breast cancer cells. (a) Unstimulated cells expressing eCFP tagged TLR9 were stained with Endoplasmatic Reticulum Tracker dye (blue, blue-white tracker). Untransfected cells were stimulated with (b) CpG-DNA (c) GpC-DNA or (d) mCpG-DNA. Cells were stained with (b-d) anti-TLR9 (red, Alexa 568) and (b, c) DAPI nucleic-acid stain. (e) Stimulated, untransfected cells were stained with anti-TLR9 (red, Alexa 568), DAPI nucleic-acid stain and anti-EEA1 an early endosome marker (green, FITC). (f) Unstimulated, untransfected cells were stained with anti-TLR9 (red, Alexa 568) and anti-EEA1 an early endosome marker (green, FITC).

    Techniques Used: Expressing, Staining, Gel Permeation Chromatography, Marker

    Colocalization analysis in BT-20 human breast cancer cells. (a) Unstimulated cells expressing eCFP tagged TLR9 were stained with Endoplasmatic Reticulum Tracker dye (blue, blue-white tracker). Untransfected cells were stimulated with (b) CpG-DNA (c) GpC-DNA or (d) mCpG-DNA. Cells were stained with (b-d) anti-TLR9 (red, Alexa 568) and (b, c) DAPI nucleic-acid stain. (e) Stimulated, untransfected cells were stained with anti-TLR9 (red, Alexa 568), DAPI nucleic-acid stain and anti-EEA1 an early endosome marker (green, FITC). (f) Unstimulated, untransfected cells were stained with anti-TLR9 (red, Alexa 568) and anti-EEA1 an early endosome marker (green, FITC).
    Figure Legend Snippet: Colocalization analysis in BT-20 human breast cancer cells. (a) Unstimulated cells expressing eCFP tagged TLR9 were stained with Endoplasmatic Reticulum Tracker dye (blue, blue-white tracker). Untransfected cells were stimulated with (b) CpG-DNA (c) GpC-DNA or (d) mCpG-DNA. Cells were stained with (b-d) anti-TLR9 (red, Alexa 568) and (b, c) DAPI nucleic-acid stain. (e) Stimulated, untransfected cells were stained with anti-TLR9 (red, Alexa 568), DAPI nucleic-acid stain and anti-EEA1 an early endosome marker (green, FITC). (f) Unstimulated, untransfected cells were stained with anti-TLR9 (red, Alexa 568) and anti-EEA1 an early endosome marker (green, FITC).

    Techniques Used: Expressing, Staining, Gel Permeation Chromatography, Marker

    37) Product Images from "Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3"

    Article Title: Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.08.013

    Alcohol- and HCV-induced phosphorylation of FOXO3. A: Western blots of Huh7.5 whole cell lysates were performed for total and phospho-Akt. Results are representative of three independent experiments. B: Whole cell lysates were prepared at indicated times (in hours) after alcohol exposure and Western blots performed for S-253 phosphorylated and total FOXO3. C: Rate of degradation of HA-FOXO3 determined by serial immunoblots after cycloheximide treatment. Numbers indicate hours after cycloheximide addition. D: Densitometry analysis of three experiments performed as in C . Data are presented as means ± SD. ∗ P
    Figure Legend Snippet: Alcohol- and HCV-induced phosphorylation of FOXO3. A: Western blots of Huh7.5 whole cell lysates were performed for total and phospho-Akt. Results are representative of three independent experiments. B: Whole cell lysates were prepared at indicated times (in hours) after alcohol exposure and Western blots performed for S-253 phosphorylated and total FOXO3. C: Rate of degradation of HA-FOXO3 determined by serial immunoblots after cycloheximide treatment. Numbers indicate hours after cycloheximide addition. D: Densitometry analysis of three experiments performed as in C . Data are presented as means ± SD. ∗ P

    Techniques Used: Western Blot

    HCV infection and alcohol alter FOXO3 in Huh7.5 cells. A: Western blots showing relative abundance of alcohol dehydrogenase in Huh7.5 cells, Huh7 cells, Huh7.5 cells with an RFP-tagged reporter protein to identify HCV infection, human liver (Hu), and mouse liver (M). B: . ∗ P
    Figure Legend Snippet: HCV infection and alcohol alter FOXO3 in Huh7.5 cells. A: Western blots showing relative abundance of alcohol dehydrogenase in Huh7.5 cells, Huh7 cells, Huh7.5 cells with an RFP-tagged reporter protein to identify HCV infection, human liver (Hu), and mouse liver (M). B: . ∗ P

    Techniques Used: Infection, Western Blot

    Effects of HCV and alcohol on FOXO3 transcriptional activity. A: Real-time RT-PCR for FOXO3-dependent genes, FOXO3, SOD2, Bim , and GADD45 , and a control, non-FOXO target gene, SOD1 , in RNA isolated from control (black bars) and 5-day HCV-infected (gray bars) Huh7.5 cells. n = 6 to 18 individual RNA preparations. P values by Wilcoxon signed-rank test for the comparison of infected and noninfected conditions were as follows: FOXO3 , P
    Figure Legend Snippet: Effects of HCV and alcohol on FOXO3 transcriptional activity. A: Real-time RT-PCR for FOXO3-dependent genes, FOXO3, SOD2, Bim , and GADD45 , and a control, non-FOXO target gene, SOD1 , in RNA isolated from control (black bars) and 5-day HCV-infected (gray bars) Huh7.5 cells. n = 6 to 18 individual RNA preparations. P values by Wilcoxon signed-rank test for the comparison of infected and noninfected conditions were as follows: FOXO3 , P

    Techniques Used: Activity Assay, Quantitative RT-PCR, Isolation, Infection

    38) Product Images from "Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation"

    Article Title: Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108662

    Effect of S1P secreted by MSCs and exogenous S1P on C2C12 and satellite cell proliferation: morphological evaluation and cell counting. A–D) Morphological evaluation of C2C12 and satellite cell proliferation by EdU incorporation assay and Ki67 expression analysis . A) Representative superimposed DIC and fluorescence images showing nuclear incorporation of EdU (green) in C2C12 cells cultured for 24 h in differentiation medium (DM) or exposed to MSC-conditioned medium obtained by culturing MSCs for 48 h in DM in the absence (MSC-DM) or in the presence of 5 µM iSK (MSC-DMiSK), and stimulated or not with 1 µM S1P (MSC-DM+S1P, MSC-DMiSK+S1P). Scale bar 50 µm. B) Representative confocal immunofluorescence images of C2C12 cells cultured in the indicated experimental conditions as in A, immunostained with antibodies against the nuclear protein Ki67 (green). Nuclei are counterstained in red with propidium iodide (PI). Yellow color indicates co-localization between red and green fluorescence signals. Scale bar 50 µm. C) Quantitative analysis of the percentage of EdU or Ki67 positive C2C12 cell nuclei expressed as percentage of the total nuclei number. D) Representative confocal immunofluorescence image of satellite cells cultured for 24 h in satellite cell proliferation medium (PM), fixed and immunostained with antibodies against Pax-7 (red) and Ki67 (green). Yellow color indicates co-localization between red and green fluorescence signals. Scale bar 50 µm. In the histogram the quantitative analysis of the percentage of Ki67 positive nuclei of satellite cells cultured for 24 h in PM or exposed to MSC-conditioned medium obtained by culturing MSCs for 48 h in PM in the absence (MSC-PM) or in the presence of 5 µM iSK (MSC-PMiSK), and stimulated or not with 1 µM S1P (MSC-PM+S1P, MSC-PMiSK+S1P). Data shown are mean±SEM and represent the results of at least three independent experiments with similar results. E) C2C12 cell counting . C2C12 cells were cultured in MSC-DM or in MSC-DMiSK and stimulated or not (vehicle) with 1 µM S1P. After 24 h of culture the cells were trypsinized and counted as described in Materials and Methods Section. The results shown are mean±SEM of at least four independent experiments performed in duplicates. Significance of difference: in C (one-way ANOVA and Newman-Keuls multiple comparison test), *p
    Figure Legend Snippet: Effect of S1P secreted by MSCs and exogenous S1P on C2C12 and satellite cell proliferation: morphological evaluation and cell counting. A–D) Morphological evaluation of C2C12 and satellite cell proliferation by EdU incorporation assay and Ki67 expression analysis . A) Representative superimposed DIC and fluorescence images showing nuclear incorporation of EdU (green) in C2C12 cells cultured for 24 h in differentiation medium (DM) or exposed to MSC-conditioned medium obtained by culturing MSCs for 48 h in DM in the absence (MSC-DM) or in the presence of 5 µM iSK (MSC-DMiSK), and stimulated or not with 1 µM S1P (MSC-DM+S1P, MSC-DMiSK+S1P). Scale bar 50 µm. B) Representative confocal immunofluorescence images of C2C12 cells cultured in the indicated experimental conditions as in A, immunostained with antibodies against the nuclear protein Ki67 (green). Nuclei are counterstained in red with propidium iodide (PI). Yellow color indicates co-localization between red and green fluorescence signals. Scale bar 50 µm. C) Quantitative analysis of the percentage of EdU or Ki67 positive C2C12 cell nuclei expressed as percentage of the total nuclei number. D) Representative confocal immunofluorescence image of satellite cells cultured for 24 h in satellite cell proliferation medium (PM), fixed and immunostained with antibodies against Pax-7 (red) and Ki67 (green). Yellow color indicates co-localization between red and green fluorescence signals. Scale bar 50 µm. In the histogram the quantitative analysis of the percentage of Ki67 positive nuclei of satellite cells cultured for 24 h in PM or exposed to MSC-conditioned medium obtained by culturing MSCs for 48 h in PM in the absence (MSC-PM) or in the presence of 5 µM iSK (MSC-PMiSK), and stimulated or not with 1 µM S1P (MSC-PM+S1P, MSC-PMiSK+S1P). Data shown are mean±SEM and represent the results of at least three independent experiments with similar results. E) C2C12 cell counting . C2C12 cells were cultured in MSC-DM or in MSC-DMiSK and stimulated or not (vehicle) with 1 µM S1P. After 24 h of culture the cells were trypsinized and counted as described in Materials and Methods Section. The results shown are mean±SEM of at least four independent experiments performed in duplicates. Significance of difference: in C (one-way ANOVA and Newman-Keuls multiple comparison test), *p

    Techniques Used: Cell Counting, Expressing, Fluorescence, Cell Culture, Immunofluorescence

    39) Product Images from "The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation"

    Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.776195

    The kindlin-2 C-terminal segment supports integrin function in HEL megakaryotic and RAW 264.7 macrophage-like cells. A , HEL cells were transiently transfected with plasmids encoding EGFP-fused K2 or its mutants and DsRed talin-H, and the extent of αIIbβ3 activation in DsRed and EGFP double-positive cells was quantified by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). The experiments were performed three times. Error bars , S.D. The total αIIbβ3 expression, measured with an mAb unaffected by the activation status of the receptor, in the presence and absence of the various K2s with or without talin-H was unaffected. The expression levels of the K2 mutants were similar. B , HEL cells were transiently transfected with plasmids encoding EGFP alone and EGFP-K2 constructs. After 24 h, EGFP expression levels in HEL cells were determined by flow cytometry; transfection efficiency was 70–80%. The transfected HEL cells were treated with PMA and allowed to adhere to fibrinogen-coated coverslips, and cell spreading was measured after 30 min. The adherent cells were fixed and stained with Alexa 568 phalloidin, and the areas of cells were measured using ImageJ software; 300 cells were quantified per construct. Error bars , S.D. ( p
    Figure Legend Snippet: The kindlin-2 C-terminal segment supports integrin function in HEL megakaryotic and RAW 264.7 macrophage-like cells. A , HEL cells were transiently transfected with plasmids encoding EGFP-fused K2 or its mutants and DsRed talin-H, and the extent of αIIbβ3 activation in DsRed and EGFP double-positive cells was quantified by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). The experiments were performed three times. Error bars , S.D. The total αIIbβ3 expression, measured with an mAb unaffected by the activation status of the receptor, in the presence and absence of the various K2s with or without talin-H was unaffected. The expression levels of the K2 mutants were similar. B , HEL cells were transiently transfected with plasmids encoding EGFP alone and EGFP-K2 constructs. After 24 h, EGFP expression levels in HEL cells were determined by flow cytometry; transfection efficiency was 70–80%. The transfected HEL cells were treated with PMA and allowed to adhere to fibrinogen-coated coverslips, and cell spreading was measured after 30 min. The adherent cells were fixed and stained with Alexa 568 phalloidin, and the areas of cells were measured using ImageJ software; 300 cells were quantified per construct. Error bars , S.D. ( p

    Techniques Used: Transfection, Activation Assay, Flow Cytometry, Cytometry, Expressing, Construct, Staining, Software

    The kindlin-2 C-terminal peptide is sufficient to influence cell spreading. αIIbβ3-CHO cells were transiently transfected with plasmids encoding PSGL-1 alone, PSGL-1–β3 (β 3 CT-containing chimera as a positive control), and K2CT chimeras: PSGL-1–K2CT, PSGL-1–K2CTY673A, PSGL-1–K2CTΔ679, and PSGL-1–K2CT double mutant. A , the expression of PSGL-1 constructs was assessed by gel electrophoresis followed by Western blotting with anti-PSGL-1 antibodies. The bottom panel shows actin as an internal loading control. B , the cells were allowed to adhere to fibrinogen-coated coverslips 12 h after transfection, and cell spreading was measured by confocal microscopy after an additional 2 h. The adherent cells were fixed and stained with the anti-PSGL-1 mAb, KPL-1 followed by Alexa Fluor 488 ( green , left ) for visualization of the expressing cells by confocal microscopy, and Alexa Fluor 568-phalloidin for actin visualization ( red , middle ), The merged images are shown on the right. Bar , 20 μm. C , the areas of PSGL-1–positive cells were measured using ImageJ software, and 100 cells were quantified. The graph is representative of three independent experiments. Error bars , S.E.
    Figure Legend Snippet: The kindlin-2 C-terminal peptide is sufficient to influence cell spreading. αIIbβ3-CHO cells were transiently transfected with plasmids encoding PSGL-1 alone, PSGL-1–β3 (β 3 CT-containing chimera as a positive control), and K2CT chimeras: PSGL-1–K2CT, PSGL-1–K2CTY673A, PSGL-1–K2CTΔ679, and PSGL-1–K2CT double mutant. A , the expression of PSGL-1 constructs was assessed by gel electrophoresis followed by Western blotting with anti-PSGL-1 antibodies. The bottom panel shows actin as an internal loading control. B , the cells were allowed to adhere to fibrinogen-coated coverslips 12 h after transfection, and cell spreading was measured by confocal microscopy after an additional 2 h. The adherent cells were fixed and stained with the anti-PSGL-1 mAb, KPL-1 followed by Alexa Fluor 488 ( green , left ) for visualization of the expressing cells by confocal microscopy, and Alexa Fluor 568-phalloidin for actin visualization ( red , middle ), The merged images are shown on the right. Bar , 20 μm. C , the areas of PSGL-1–positive cells were measured using ImageJ software, and 100 cells were quantified. The graph is representative of three independent experiments. Error bars , S.E.

    Techniques Used: Transfection, Positive Control, Mutagenesis, Expressing, Construct, Nucleic Acid Electrophoresis, Western Blot, Confocal Microscopy, Staining, Software

    40) Product Images from "Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking"

    Article Title: Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02904-x

    LSP1 is a component of the podosome cap structure. a – d Confocal micrographs of a macrophage stained for LSP1 using specific primary antibody and Alexa 488-labeled secondary antibody ( a , green), for F-actin using Alexa405-labeled phalloidin ( b , red), and for vinculin using specific primary antibody and Alexa568-labeled secondary antibody ( c , white), with merge ( d ). White boxes in a – d indicate detail images shown as insets. Dashed boxes in insets indicate single podosome shown in x – z section in a ′– d ′ and in x – y section in a ″– d ″ with respective cross-section planes shown as white dotted lines. Dashed yellow lines in d ′ indicate confocal planes used for measurements of respective fluorescence intensities shown in e . Note the cap-like localization of LSP1 on top of the F-actin core (1), which can appear ring-like in a lower optical section (2) Scale bars: 5 µm in a – d , 1 µm in insets, 0.5 µm in a ″– d ″. f . g Domain structure of LSP1 full length and deletion mutants. LSP1 features an acidic N-terminal half containing a hypothetical Ca 2+ binding domain, two caldesmon-like F-actin binding domains (C1, C2) and two villin-headpiece-like F-actin binding domains (V1, V2). First and last amino acid residues are indicated. “+” and “−” indicate the presence or absence of the respective construct at podosomes
    Figure Legend Snippet: LSP1 is a component of the podosome cap structure. a – d Confocal micrographs of a macrophage stained for LSP1 using specific primary antibody and Alexa 488-labeled secondary antibody ( a , green), for F-actin using Alexa405-labeled phalloidin ( b , red), and for vinculin using specific primary antibody and Alexa568-labeled secondary antibody ( c , white), with merge ( d ). White boxes in a – d indicate detail images shown as insets. Dashed boxes in insets indicate single podosome shown in x – z section in a ′– d ′ and in x – y section in a ″– d ″ with respective cross-section planes shown as white dotted lines. Dashed yellow lines in d ′ indicate confocal planes used for measurements of respective fluorescence intensities shown in e . Note the cap-like localization of LSP1 on top of the F-actin core (1), which can appear ring-like in a lower optical section (2) Scale bars: 5 µm in a – d , 1 µm in insets, 0.5 µm in a ″– d ″. f . g Domain structure of LSP1 full length and deletion mutants. LSP1 features an acidic N-terminal half containing a hypothetical Ca 2+ binding domain, two caldesmon-like F-actin binding domains (C1, C2) and two villin-headpiece-like F-actin binding domains (V1, V2). First and last amino acid residues are indicated. “+” and “−” indicate the presence or absence of the respective construct at podosomes

    Techniques Used: Staining, Labeling, Fluorescence, Binding Assay, Construct

    LSP1 interacts with myosin IIA and regulates its recruitment to podosomes. a , b Confocal micrographs of mixed populations of macrophages, stained for myosin IIA using specific primary antibody ( a ) or for F-actin using Alexa-405-labeled phalloidin ( b ). Cells treated with LSP1 siRNA express GFP, cells treated with control siRNA express mCherry, as shown in inset in a . c , d ImageJ-based macros were used to identify myosin IIA at podosomes ( c ) and F-actin-rich podosome cores ( d ). Scale bar: 10 µm. e – h Statistical evaluation of ( e ) myosin-based fluorescence at podosomes, ( f ) F-actin-based fluorescence at podosomes and size of areas analysed for podosomal myosin IIA ( g ) and F-actin ( h ) podosome-covered area. Each dot represents the mean intensity of all the individual podosomes detected in a single cell (~500 podosomes/cell on average), five cells from three different donors. Values are given as mean ± SD. * P
    Figure Legend Snippet: LSP1 interacts with myosin IIA and regulates its recruitment to podosomes. a , b Confocal micrographs of mixed populations of macrophages, stained for myosin IIA using specific primary antibody ( a ) or for F-actin using Alexa-405-labeled phalloidin ( b ). Cells treated with LSP1 siRNA express GFP, cells treated with control siRNA express mCherry, as shown in inset in a . c , d ImageJ-based macros were used to identify myosin IIA at podosomes ( c ) and F-actin-rich podosome cores ( d ). Scale bar: 10 µm. e – h Statistical evaluation of ( e ) myosin-based fluorescence at podosomes, ( f ) F-actin-based fluorescence at podosomes and size of areas analysed for podosomal myosin IIA ( g ) and F-actin ( h ) podosome-covered area. Each dot represents the mean intensity of all the individual podosomes detected in a single cell (~500 podosomes/cell on average), five cells from three different donors. Values are given as mean ± SD. * P

    Techniques Used: Staining, Labeling, Fluorescence

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    Article Snippet: .. Annexin V, conjugated to Alexa Fluor-568, was used as an additional marker for PS exposure (Life Technologies, #A13202, 1:250). .. Apoptotic photoreceptors were labeled with a terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay (Roche, Indianapolis, IN) according to the manufacturer’s specifications.

    Staining:

    Article Title: An ancestral TMEM16 homolog from Dictyostelium discoideum forms a scramblase
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    Article Title: Distinct modes of cell competition shape mammalian tissue morphogenesis
    Article Snippet: .. To obtain samples required to generate our cell competition signature, K14-H2B-GFP cells were further stained for AnnexinV (Alexa Fluor 568 conjugated; ThermoFisher Scientific, 1:200); 2 96-well plates of single cells were obtained for each lin-/GFP+ /AnnexinV+ (losers) and 2 plates worth of lin-/GFP+ AnnexinV– (winners). ..

    Article Title: Antiproliferative Activity of Cyanophora paradoxa Pigments in Melanoma, Breast and Lung Cancer Cells
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    Article Title: Distinct modes of cell competition shape mammalian tissue morphogenesis
    Article Snippet: .. Single cell suspensions were stained with anti-CD49f/alpha6-PE/Cy7 (Biolegend, 1:1000) and/or AnnexinV (Alexa Fluor 568 conjugated, Life Technologies, 1:200) and sorted using a BD FACSAriaII. .. RNA was isolated in Trizol from alpha6hi, RFP+, lin- cells using the Direct-zol RNA Miniprep Plus kit (Zymo Research).

    Binding Assay:

    Article Title: An ancestral TMEM16 homolog from Dictyostelium discoideum forms a scramblase
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    Translocation Assay:

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    Labeling:

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    Thermo Fisher goat anti rabbit igg alexa fluor 568
    Goat Anti Rabbit Igg Alexa Fluor 568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg alexafluor 568 conjugated secondary antibody
    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with <t>AlexaFluor-568</t> phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p
    Goat Anti Rabbit Igg Alexafluor 568 Conjugated Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg conjugated to alexa fluor 568
    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with <t>AlexaFluor-568</t> phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p
    Goat Anti Rabbit Igg Conjugated To Alexa Fluor 568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with AlexaFluor-568 phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with AlexaFluor-568 phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Generated, Two Tailed Test

    Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Staining, Migration, Generated, Knock-Out

    Representative images of wild-type and twinfilin knockout B16-F1 cells. (A) B16-F1 cells were stained with AlexaFluor-568 phalloidin (F-actin) and anti-p34 antibody (the Arp2/3 complex). Scale bars = 10 μ M. (B) Mean F-actin intensity in B16-F1 wild-type and twinfilin knockout cells. Number of measured cells were: B16-F1 wt = 1,958, twf1-KO-g1 = 1,045, twf2-KO-g3#1 = 1,285, twf1/2-KO-g3 = 1,265, twf1/2-KO-g4 = 1,707. (C) Mean F-actin intensity in the Arp2/3 complex positive regions of B16-F1 wild-type and knockout cells. The Arp2/3 complex positive regions were identified based on p34-antibody staining. Number of measured cells were: B16-F1 wt = 1,658, twf1-KO-g1 = 884, twf2-KO-g3#1 = 1,009, twf1/2-KO-g3 = 1,100, twf1/2-KO-g4 = 1157. Statistical significances in panels B and C were calculated with Mann-Whitney two-tailed test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Representative images of wild-type and twinfilin knockout B16-F1 cells. (A) B16-F1 cells were stained with AlexaFluor-568 phalloidin (F-actin) and anti-p34 antibody (the Arp2/3 complex). Scale bars = 10 μ M. (B) Mean F-actin intensity in B16-F1 wild-type and twinfilin knockout cells. Number of measured cells were: B16-F1 wt = 1,958, twf1-KO-g1 = 1,045, twf2-KO-g3#1 = 1,285, twf1/2-KO-g3 = 1,265, twf1/2-KO-g4 = 1,707. (C) Mean F-actin intensity in the Arp2/3 complex positive regions of B16-F1 wild-type and knockout cells. The Arp2/3 complex positive regions were identified based on p34-antibody staining. Number of measured cells were: B16-F1 wt = 1,658, twf1-KO-g1 = 884, twf2-KO-g3#1 = 1,009, twf1/2-KO-g3 = 1,100, twf1/2-KO-g4 = 1157. Statistical significances in panels B and C were calculated with Mann-Whitney two-tailed test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Knock-Out, Staining, MANN-WHITNEY, Two Tailed Test

    Knockout of twinfilins leads to abnormal F-actin accumulation in lamellipodia and perinuclear region. (A) Representative images of wild-type and twf1/twf2-KO mouse B16-F1 cells stained with AlexaFluor-568 phalloidin and anti-p34 antibody to visualize F-actin and the Arp2/3 complex, respectively. Scale bar = 10 μ m. (B) F-actin intensities in the cytoplasmic regions of wild-type, twf1/twf2-KO, and knockout cells expressing EGFP-TWF-1 measured by high-content image analysis. Number of cells analyzed were: B16-F1 wt = 4,875, twf1/twf2-KO-g3 = 5,731, twf1/twf2-KO-g3 + EGFP-TWF-1 = 197. (C) Lamellipodia protrusion velocities of wild-type, twf1/twf-2 knockout, and knockout cells expressing EGFP-TWF-1. Data represent individual cells with mean and standard deviations shown. Statistical significances in panels B and D were calculated with Mann-Whitney two-tailed test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Knockout of twinfilins leads to abnormal F-actin accumulation in lamellipodia and perinuclear region. (A) Representative images of wild-type and twf1/twf2-KO mouse B16-F1 cells stained with AlexaFluor-568 phalloidin and anti-p34 antibody to visualize F-actin and the Arp2/3 complex, respectively. Scale bar = 10 μ m. (B) F-actin intensities in the cytoplasmic regions of wild-type, twf1/twf2-KO, and knockout cells expressing EGFP-TWF-1 measured by high-content image analysis. Number of cells analyzed were: B16-F1 wt = 4,875, twf1/twf2-KO-g3 = 5,731, twf1/twf2-KO-g3 + EGFP-TWF-1 = 197. (C) Lamellipodia protrusion velocities of wild-type, twf1/twf-2 knockout, and knockout cells expressing EGFP-TWF-1. Data represent individual cells with mean and standard deviations shown. Statistical significances in panels B and D were calculated with Mann-Whitney two-tailed test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Knock-Out, Staining, Expressing, MANN-WHITNEY, Two Tailed Test