Journal: Journal of gastroenterology

Article Title: Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium.

doi: 10.1007/s00535-020-01695-7

Figure Lengend Snippet: Fig. 1 Induction of EECs from hiPSCs. a A schematic diagram of the experiment to examine the conditions underlying the differentiation of foregut (FG) into dorsal anterior foregut (dAFG). b An expression analysis of SOX2 and p63 at Day 13 by semi-quantitative RT-PCR. GAPDH was used as an endogenous control. c Immunostaining of SOX2 (green), p63 (red) and Hoechst (blue) at Day 13. Representative fluorescence is shown. Scale bars, 50 lm. d A schematic diagram of the experiment to examine the conditions underlying the differentiation of dAFG into EECs. e Expression analyses of p63, SOX2, CK13, CK5 and PAX9 at Day 21 by semi-quantitative RT-PCR. GAPDH was used as an endogenous control. Total RNA of human normal esophageal tissue was used as a positive control

Article Snippet: The primary antibodies were rabbit anti-SOX2 (ab97959; abcam), rabbit anti-CK13 (ab92551, dilution 1:200; abcam), goat anti-p63 (BAF1916, dilution 1:200; R&D Systems), rat anti-PAX9 (ab28538, dilution 1:200; abcam), goat anti-Ecadherin (AF648, dilution 1:200; R&D Systems), rabbit anti-CK5 (GTX113219, dilution 1:200; GeneTex, Irvine, CA, USA), rabbit anti-Claudin4 (ab210796, dilution 1:200; abcam) and rabbit anti-Involucrin (ab53112, dilution 1:200; abcam).

Techniques: Expressing, Quantitative RT-PCR, Control, Immunostaining, Positive Control

Journal: Journal of gastroenterology

Article Title: Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium.

doi: 10.1007/s00535-020-01695-7

Figure Lengend Snippet: Fig. 2 The expression of esophageal epithelial marker proteins in the hiPSC-derived cells and fetal mouse esophagus at E17.5. a A schematic representation of the stepwise differentiation protocol and marker genes at each step. b Immunostaining of the SOX2, p63, CK5, E-cadherin, CK13 and PAX9 at Day 24 in the derivatives of hiPSCs (upper panels) and fetal mouse esophagus at E17.5 (lower panels). Scale bars, 20 lm

Article Snippet: The primary antibodies were rabbit anti-SOX2 (ab97959; abcam), rabbit anti-CK13 (ab92551, dilution 1:200; abcam), goat anti-p63 (BAF1916, dilution 1:200; R&D Systems), rat anti-PAX9 (ab28538, dilution 1:200; abcam), goat anti-Ecadherin (AF648, dilution 1:200; R&D Systems), rabbit anti-CK5 (GTX113219, dilution 1:200; GeneTex, Irvine, CA, USA), rabbit anti-Claudin4 (ab210796, dilution 1:200; abcam) and rabbit anti-Involucrin (ab53112, dilution 1:200; abcam).

Techniques: Expressing, Marker, Derivative Assay, Immunostaining

Journal: Journal of gastroenterology

Article Title: Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium.

doi: 10.1007/s00535-020-01695-7

Figure Lengend Snippet: Fig. 5 Treatment with an RARc-specific agonist instead of ATRA also induced stratified layers of cells expressing esophageal epithelial markers. a A schematic diagram of the EEC differentiation protocol with an RARc-specific agonist. b Immunostaining of SOX2, p63, CK5, E-cadherin, CK13 and PAX9 in the derivatives of hiPSCs. Scale bars, 20 lm

Article Snippet: The primary antibodies were rabbit anti-SOX2 (ab97959; abcam), rabbit anti-CK13 (ab92551, dilution 1:200; abcam), goat anti-p63 (BAF1916, dilution 1:200; R&D Systems), rat anti-PAX9 (ab28538, dilution 1:200; abcam), goat anti-Ecadherin (AF648, dilution 1:200; R&D Systems), rabbit anti-CK5 (GTX113219, dilution 1:200; GeneTex, Irvine, CA, USA), rabbit anti-Claudin4 (ab210796, dilution 1:200; abcam) and rabbit anti-Involucrin (ab53112, dilution 1:200; abcam).

Techniques: Expressing, Immunostaining

Journal: Scientific reports

Article Title: Assessment of the toxic effect of benzalkonium chloride on human limbal stem cells.

doi: 10.1038/s41598-025-96919-2

Figure Lengend Snippet: Fig. 3. (A) Agarose gel electropherogram of amplification products (passage 1). Lane 1: DNA ladder (M), lanes 2–3: KRT3 (101 bp), lanes 4–5: KRT12 (190 bp), lane 6: DNA ladder (M), lanes 7–8: TP63 (96 bp), lanes 9–10: ABCG2 (144 bp), lanes 11–12: ACTβ (131 bp), lane 13: DNA ladder (M). NC, negative control. (B) Visualization of lack of expression of CK3, and CK12 and expression of positive markers p63 and ABCG2, in LSCs cultured in a medium supplemented with H/I/E. FITC-conjugated specific antibodies (green); DAPI- stained cell nuclei (blue). IC, isotype control. Scale bars = 15 μm.

Article Snippet: After rinsing with PBS, the cells were incubated with blocking buffer for 1 h, rinsed again, and labelled with rabbit anti-ABCG2 polyclonal (1:50; 27286-AP; Proteintech, USA), rabbit anti-p63 polyclonal (1:50; 12143-1-AP; Proteintech, USA), mouse anti-CK3 monoclonal (1:50; ab68260; Abcam, UK), and rabbit anti-CK12 monoclonal (1:50; ab185627; Abcam, UK) primary antibodies at 4 °C overnight.

Techniques: Agarose Gel Electrophoresis, Amplification, Negative Control, Expressing, Cell Culture, Staining, Control

Journal: bioRxiv

Article Title: Heterogeneous potential of human airway basal cells: Holoclone-stem cell identification in a clinical-grade system

doi: 10.1101/2023.12.28.573530

Figure Lengend Snippet: (A) IF staining of AT (left) and AB (right) cultures at four different time points of airway epithelial cell lifespan (representative images of n = 3 AT and n = 3 AB primary human independent strains). Scale bars, 50 μm. (B) WB analysis on total cell extracts from six expansion passages (AT2 strain) representative of five consecutive lifespan intervals, immunostained with indicated antibodies. Experiment conducted on n = 3 AT strains (n = 5 technical replicates) and n = 3 AB strains (n = 4 technical replicates). (C) Histograms showing the quantification of the expression levels from top left to bottom right of CK14, Involucrin, p63α, BMI1 (all normalized per GAPDH) and SOX2 (normalized per Vinculin). Average and SD of n = 3 AT and n = 3 AB displayed per each time range (see “Star Methods”). Independent strains are indicated with different shapes. Unpaired, biparametric, two-tailed t test *p < 0.05.; ** p < 0.01 and *** p < 0.001.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p63-alpha (1:5000, customized), rabbit monoclonal anti-BMI1 (1:500, Cell Signalling Technology), rabbit monoclonal anti-SOX2 (1:200, Cell Signalling Technology), mouse monoclonal anti-GAPDH (1:10.000, Abcam), mouse monoclonal anti-Vinculin (1:10.000, Sigma-Aldrich), rabbit polyclonal anti-KERATIN 14 (1:40.000, Biolegend), mouse monoclonal anti-Involucrin (1:10.000, Leica Microsystems), mouse monoclonal anti-Actin (1:5000, Abcam).

Techniques: Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Heterogeneous potential of human airway basal cells: Holoclone-stem cell identification in a clinical-grade system

doi: 10.1101/2023.12.28.573530

Figure Lengend Snippet: (A) Violin plot showing the size measured in mm of AT (left) and AB (right) different clones. AT analysed clones: H, n = 36; EM, n = 86; IM, n = 112; LM, n = 56; P, n = 45 belonging to n = 3 independent AT strains; AB analysed clones: H, n = 35; EM, n = 44; IM, n = 52; LM, n = 13; P, n = 9 belonging to n = 2 independent AB strains. Dots represent single clones. Median, first and third quartiles are displayed. (B) WB analysis on total cell extracts from the progeny of AT2 clones (H, n = 3; EM, n = 2; IM, n = 1; LM, n = 2) immunostained with indicated antibodies (image representative of n = 2 analysis conducted on independent clones). Due to their very limited residual proliferative potential, the amount of material collected by AT2 paraclone’s progenies was not sufficient to carry out a reliable WB analysis. (C) From left to right, histograms showing the quantification of the expression levels of p63α, BMI1 (normalized per GAPDH) and SOX2 (normalized per GAPDH in AT and Actin in AB). Average and SD displayed per each clonal category (see “Star Methods”). AT2 analysed clones: H, n = 3; EM, n = 5; IM, n = 5; LM, n = 3. AB1 analysed clones: H, n = 3; EM, n = 2; IM, n = 3; LM, n = 3. Unpaired, biparametric, two-tailed t test *p < 0.05.; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p63-alpha (1:5000, customized), rabbit monoclonal anti-BMI1 (1:500, Cell Signalling Technology), rabbit monoclonal anti-SOX2 (1:200, Cell Signalling Technology), mouse monoclonal anti-GAPDH (1:10.000, Abcam), mouse monoclonal anti-Vinculin (1:10.000, Sigma-Aldrich), rabbit polyclonal anti-KERATIN 14 (1:40.000, Biolegend), mouse monoclonal anti-Involucrin (1:10.000, Leica Microsystems), mouse monoclonal anti-Actin (1:5000, Abcam).

Techniques: Clone Assay, Expressing, Two Tailed Test

Journal: Veterinary Ophthalmology

Article Title: Morphological and immunohistochemical characteristics of the equine corneal epithelium

doi: 10.1111/vop.12651

Figure Lengend Snippet: Sources, dilutions, fixations and pretreatments of the antibodies used

Article Snippet: p63 , Potential SC marker , Polyclonal orb214808 , Rabbit , Biorbyt, Cambridge, UK , 1:2500 , f , 0.01 mol/L citrate buffer pH 6.0, 30 min steamer.

Techniques: Cell Differentiation, Marker

Journal: Veterinary Ophthalmology

Article Title: Morphological and immunohistochemical characteristics of the equine corneal epithelium

doi: 10.1111/vop.12651

Figure Lengend Snippet: Semiquantitative analysis of immunohistochemical staining for cytoskeletal proteins, proliferation, differentiation, and stem cell markers. CK 14, p63, and NGF showed a gradual decrease from crypt to center. Statistically significant age differences (*, P < 0.05) were detected in the limbal zone for NGF and in the noncrypt zone for ABCG2. Bars represent mean values from six horses of both age groups and whiskers represent SD.

Article Snippet: p63 , Potential SC marker , Polyclonal orb214808 , Rabbit , Biorbyt, Cambridge, UK , 1:2500 , f , 0.01 mol/L citrate buffer pH 6.0, 30 min steamer.

Techniques: Immunohistochemical staining, Staining

Journal: Veterinary Ophthalmology

Article Title: Morphological and immunohistochemical characteristics of the equine corneal epithelium

doi: 10.1111/vop.12651

Figure Lengend Snippet: Immunohistochemical staining of proposed SC‐associated markers Ki67, p63, NGF, ABCG2, and EGFR. Representative pictures of crypts, limbus, and central corneal epithelium are shown for both foals (left panels) and adult horses (right panels). Potential stem cell markers (p63, NGF, and ABCG2) were present in every examined region with decreasing frequency toward the center. Scale bar =30 µm

Article Snippet: p63 , Potential SC marker , Polyclonal orb214808 , Rabbit , Biorbyt, Cambridge, UK , 1:2500 , f , 0.01 mol/L citrate buffer pH 6.0, 30 min steamer.

Techniques: Immunohistochemical staining, Staining

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: Analysis of composition of autophagy complexes by immunoprecipitation (IP) studies. A: protein blots of cellular lysates (n = 3) from differentiated vector- (V-) and G0-podocytes were probed for ATG14L and reprobed for UVRAG, beclin-1, PI3KC3 (VPS34), APOL1, and GAPDH. Representative gels are displayed. i: Distribution densitometric data of ATG14L/GAPDH ratios from A are shown in a dot plot. ii: Distribution densitometric data of UVRAG/GAPDH ratios from A are shown in a dot plot. iii: Distribution densitometric data of beclin-1/GAPDH ratios from A are shown in a dot plot. iv: Distribution densitometric data of PI3KC3/GAPDH ratios from A are shown in a dot plot. v: Distribution densitometric data of APOL1/GAPDH ratios from A are shown in a dot plot. *P < 0.05 vs. V. B: cellular lysates were immunoprecipitated with an anti-ATG14L antibody (n = 6). Protein blots of IP fractions were probed for ATG14L and reprobed for beclin-1, PI3KC3, and APOL1. IgG expression was used as a loading marker. Gels from three different IP fractions are displayed. i: Distribution densitometric data of ATG14L/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V. ii: Distribution densitometric data of beclin-1/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V. iii: Distribution densitometric data of PI3KC3/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V; iv: Distribution densitometric data of APOL1/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V. C: cellular lysates of V- and G0-podocytes from A were immunoprecipitated with anti-UVRAG antibody (n = 6). Protein blots were probed for UVRAG, beclin-1, PI3KC3, APOL1, and IgG. Gels from three independent IP fractions are shown. i: Distribution densitometric data of UVRAG/IgG ratios from C are shown in a dot plot. *P < 0.05 vs. V. ii: Distribution densitometric data of beclin-1/IgG ratios from C are shown in a dot plot. *P < 0.05 vs. V. iii: Distribution densitometric data of PI3KC3/IgG ratios from 3C are shown in a dot plot. *P < 0.05 vs. V. iv: Distribution densitometric data of APOL1/IgG ratios from C are shown in a dot plot. *P < 0.05 vs. V. D: protein blots of cellular lysates of V- and G0-podocytes were probed for APOL1, PI3KC3, and beclin-1 and reprobed for actin (n = 6). Cellular lysates from three independent lysates are shown. i: Distribution densitometric data of APOL1/actin ratios from D are shown in a dot plot. *P < 0.05 vs. V. ii: Distribution densitometric data of PI3KC3/actin ratios from D are shown in a dot plot. *P < 0.05 vs. V. iii: Distribution densitometric data of beclin-1/actin ratios from D are shown in a dot plot. *P < 0.05 vs. V. E: cellular lysates of V- and G0-podocytes were immunoprecipitated with anti-APOL1 antibody. IP fractions were probed for APOL1, PI3KC3, beclin-1, and IgG. Gels from three independent IP fractions are displayed. i: Distribution densitometric data of APOL1/IgG in ratios from E are shown in a dot plot. *P < 0.05 vs. V. ii: Distribution densitometric data of PI3KC3/IgG ratios from 3E are shown in a dot plot. iii: Distribution densitometric data of beclin-1/IgG ratios from E are shown in a dot plot. *P < 0.05 vs. V. APOL1, apolipoprotein L1; PI3KC3, class III phosphatidylinositol 3-kinase; ATG14L, autophagy-related gene 14L; UVRAG, UV radiation resistance-associated gene.

Article Snippet: Immunoprecipitation Studies Lysates from V- and G0-podocytes were first immunoprecipitated following the addition of 5 μg of anti-APOL1 (Proteintech), 5 μg of anti-ATG14L (Abcam), 5 μg of anti-Rubicon (Sana Cruz Biotechnology), or 5 μg of anti-UVRAG (Proteintech) antibodies.

Techniques: Immunoprecipitation, Plasmid Preparation, Expressing, Marker

Journal: American Journal of Physiology - Cell Physiology

Article Title: Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu

doi: 10.1152/ajpcell.00538.2018

Figure Lengend Snippet: APOL1 risk milieus induce Rubicon complex formation in podocytes. A: protein blots of cellular lysates of vector- (V-), G0-, G1-, and G2-podocytes were probed for UVRAG, Rubicon, PI3KC3,and APOL1 and reprobed for GAPDH. Gels from three independent lysates are displayed. i: Distribution densitometric data of UVRAG/GAPDH ratios from A are shown in a dot plot. ii: Distribution densitometric data of Rubicon/GAPDH ratios from A are shown in a dot plot. *P < 0.05 vs. G0. iii: Distribution densitometric data of PI3KC3/GAPDH ratios from A are shown in a dot plot. *P < 0.05 vs. G0. iv: distribution densitometric data of APOL1/GAPDH ratios from A are shown in a dot plot. *P < 0.05 compared vs. V. B: cellular lysates of V-, G0-, G1-, and G2-podocytes from 4A were immunoprecipitated with an anti-Rubicon antibody. Immunoprecipitation (IP) fractions were probed for UVRAG, Rubicon, PI3KC3, and APOL1. IgG was used as a loading marker. Gels from three independent IP fractions are displayed. i: Distribution densitometric data of UVRAG/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V and G0. ii: Distribution densitometric data of Rubicon/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V and G0. iii: Distribution densitometric data of PI3KC3/IgG ratios from B are shown in a dot plot. iv: Distribution densitometric data of APOL1/IgG ratios from B are shown in a dot plot. *P < 0.05 vs. V. C: RNAs were extracted from different sets of V-, G0-, G1-, and G2-podocytes (n = 5). cDNAs were amplified with a specific primer for Rubicon. Cumulative data are shown in a dot plot. ***P < 0.001 vs. V and G. APOL1, apolipoprotein L1; PI3KC3, class III phosphatidylinositol 3-kinase; Rubicon, run domain beclin-1-interacting and cysteine-r UVRAG, UV radiation resistance-associated gene.

Article Snippet: Immunoprecipitation Studies Lysates from V- and G0-podocytes were first immunoprecipitated following the addition of 5 μg of anti-APOL1 (Proteintech), 5 μg of anti-ATG14L (Abcam), 5 μg of anti-Rubicon (Sana Cruz Biotechnology), or 5 μg of anti-UVRAG (Proteintech) antibodies.

Techniques: Plasmid Preparation, Immunoprecipitation, Marker, Amplification

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were infected with WSN or VIC at a MOI of 5 PFU/cell for 8 h, or mock-infected. Fixed cells were stained for the viral NP and the cellular markers for ER sheets and ER tubules, CLIMP63 and RTN3, respectively. The RTN3 staining was used to delineate the cell edges. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10 µm. B. A549 cells treated as in A were analyzed with the Cell Profiler software in order to segment CLIMP63+ and RTN3+ areas based on the Otsu thresholding method. The ratios of the CLIMP63+ area to the RTN3+ area are shown. Each dot represents one cell, and the data from three independent experiments are shown (black, grey and white dots). The median and interquartile values are represented as box-plots (200-350 cells per condition). *** : p-value < 0.001, one-way ANOVA. C-D. A549 cells were infected or mock-infected as in A. At 8 hpi total cell lysates were prepared and analysed by western blot, using the indicated antibodies. (C) Cropped blots of one representative experiment out of three are shown. (D) The signals for CLIMP63 and RTN3 are normalised over the tubulin signal and expressed as percentages (100% : mock-infected cells). The data shown are the mean ± SD of three independent experiments. No significant difference is detected between infected and mock-infected cells (two-way ANOVA with Sidak’s multiple comparison test).

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Staining, Microscopy, Software, Western Blot, Comparison

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were infected with WSN at a MOI of 5 PFU/cell for 8 h. Fixed cells were stained for the viral HA and cellular CLIMP63 proteins. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10µm. B. Fluorescence intensity profile for HA (cyan) and CLIMP63 (magenta) along the white line drawn in panel A (merge inset), starting from the knob. C. A549 cells were infected with ZIKV PF13 at a MOI of 5 PFU/cell for 24 h, or mock-infected. Fixed cells were stained for the viral NP and the cellular RTN3 and CLIMP63 proteins. The RTN3 staining was used to delineate the cell edges. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. White arrows indicate viral factories surrounded with remodelled ER membranes. Scale bar: 10µm.

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Staining, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549cells were infected with the WSN-PB2-Strep virus at a MOI 5 PFU/cell for 8 h. Fixed cells were stained for NP, PB2 (StrepTactin-488) and RAB11. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10µm. B. Fluorescence intensity profile for NP (red), PB2-Strep-tag (cyan) and RAB11 (magenta) along the white line drawn in panel A (merge inset), starting from the knob. C. A549 cells were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 8 h, or mock infected. Fixed cells were stained for the viral NP and cellular RTN3 and CLIMP63 proteins. The difference in permeabilisation protocols (saponin versus Triton) most likely accounts for the difference in NP signal patterns in this experiment compared to the experiment shown in Figure 2A. Scale bar: 10µm.

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Virus, Staining, Microscopy, Fluorescence, Strep-tag, Control

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 4 h. Fixed cells were stained for the viral NP and the cellular RAB11 and CLIMP63 proteins. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. White stars: non-specific nuclear staining enhanced upon siRNA treatment. Scale bar: 10µm. B. Fluorescence intensity profiles for NP (red) and CLIMP63 (magenta) along the white lines drawn in panel A (merge insets), starting from the knob. C. U2OS cells stably expressing the ER translocon Sec61ß fused to mEmerald (U2OS-Sec61ß-mEmerald) were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 8 h. Fixed cells were stained for NP and Sec61ß-mEmerald (anti-GFP antibody) and images were acquired using STED microscopy. Scale bar: 10µm. D. U2OS-Sec61ß-mEmerald cells treated as in A were analyzed to assess colocalization of NP and Sec61ß, using a pixel-based method to determine the Pearson coefficient on a region of interest corresponding to the whole cell or to the perinuclear region, as defined in the Methods section. Each dot represents one cell, and the data from three independent experiments are shown (black, grey and white dots. The median and interquartile values are represented as box-plots (18 and 19 cells treated with the NT or RAB11A-specific siRNAs, respectively). ns: non-significant (unpaired t-test).

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Control, Infection, Staining, Microscopy, Fluorescence, Stable Transfection, Expressing