Journal: PLoS ONE

Article Title: Targeted Mutation of the Mouse Grp94 Gene Disrupts Development and Perturbs Endoplasmic Reticulum Stress Signaling

doi: 10.1371/journal.pone.0010852

Figure Lengend Snippet: (A) Representative Western blot results on effect of GRP94 depletion on cell proliferation and apoptosis. Grp94 +/+ and −/− ESCs were treated with 300 nM Tg for the indicated time (hr). The whole cell lysates were subjected to Western blot with antibodies against PCNA, caspase-7 (C-7) and β-actin. Both pro C-7 (the uncleaved form) and cleaved C-7 (active form) were detected by the anti-C-7 antibody. The cleaved C-7 protein level was quantitated and normalized against β-actin. The level of cleaved C-7 in Grp94 +/+ ESCs treated with Tg for 4 hr was set as 1. (B) The same as in (A) with 1.5 µg/ml Tunicamycin (Tu) treated cells. The Western blots were performed with antibodies against cleaved caspase-3 (C-3) and β-actin. The cleaved C-3 protein level was quantitated and normalized against β-actin. The level of cleaved C-3 in Grp94 +/+ ESCs without Tu treatment was set as 1. These experiments were repeated two times.

Article Snippet: The primary antibodies used included the following: mouse anti-KDEL(10C3) (1∶1000), rat anti-GRP94 (1∶2500), rabbit anti-calnexin (1∶2000), and rabbit anti-calreticulin (1∶2000) from Stressgen; goat anti-GRP78(C20) (1∶5000), rabbit anti-PERK (H-300) (1∶500), rabbit anti-ATF4 (CREB-2) (1∶1000), goat anti-HSP70 (K-20) (1∶1000), mouse anti-PCNA (PC10) (1∶1000), mouse anti-CHOP (1∶1000), goat anti-EDEM (1∶500) and rabbit anti-XBP1 (1∶500) from Santa Cruz Biotechnology; rabbit anti-phospho-PERK (Thr980) (1∶500), mouse anti-cleaved-caspase-7 (Asp198), rabbit anti-phospho-eIF2α (1∶1000), rabbit anti- eIF2α (1∶1000) and rabbit anti-cleaved caspase-3 (Asp175) (1∶500) from Cell Signaling; rabbit anti-PDI (1∶2000) from Assay Designs; mouse anti-β-actin (1∶5000) from Sigma-Aldrich; and rabbit anti-p62 (SQSTM1) (1∶1000) from BIOMOL.

Techniques: Western Blot

Journal: Cancers

Article Title: CLDN1 Sensitizes Triple-Negative Breast Cancer Cells to Chemotherapy

doi: 10.3390/cancers14205026

Figure Lengend Snippet: “Claudin-1-high” TNBC cells are more sensitive than “claudin-1-low” TNBC cells to chemotherapy. HCC1806, MDA-MB-231 and Hs578T cells were treated for 72 h with increasing doses of ( A ) 5-FU (0, 5, 10, 25, 50, 100, 200 μM), ( B ) PTX (0, 1, 2.5, 5, 10, 15, 20, 25 nM) and ( C ) DOX (0, 5, 10, 20, 30, 50, 160, 400, 800 nM). Cell viability was determined by measuring ATP level. The results are expressed as a percentage of the control cells exposed to the solvent and IC50s are specified above the graphs. Cells were treated for 72 h at different concentrations of ( D ) 5-FU (0, 25, 50, 100, 150, 200 μM); ( E ) PTX (0, 1, 2.5, 5, 18 nM); and ( F ) DOX (0, 10, 25, 50, 150, 400 nM). Analysis of CLDN1, cleaved apoptotic markers PARP-1 (cPARP) and caspase 7 (cCASP7) expression was performed by western blot. The GAPDH was used as an internal reference. Western Blots expression are representative of 3 to 4 independent experiments. The values represent the means ± SEM of three to four different experiments. A p -value of less than 0.05 was considered statistically significant with * p < 0.05. The uncropped blots are shown in .

Article Snippet: The anti-cleaved caspase 7 antibody (#9494S, Cell Signaling Technology, Saint-Cyr-L’École, France) was diluted at 1:800, the anti-cleaved PARP-1 antibodies (552596, BD Biosciences, Le Pont de Claix, France) and anti-CLDN1 (C5838-02C, US Biological, Nanterre, France) were diluted at 1:1000 and the GAPDH antibody (60004-1-IgG, ProteinTech, Illkirch-Graffenstaden, France) was diluted at 1:5000.

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: CLDN1 Sensitizes Triple-Negative Breast Cancer Cells to Chemotherapy

doi: 10.3390/cancers14205026

Figure Lengend Snippet: CLDN1 is involved in the chemosensitivity of HCC1806 “claudin-1-high” cells to 5-FU, PTX and DOX. ( A ) HCC1806 cells were transfected with siRNA directed against CLDN1 (siCLDN1) or with a scramble siRNA (scRNA) and CLDN1 expression was studied by western blot. 7 h after transfection, cells were treated for 72 h with ( B – D ) 50 μM of 5-FU, ( E – G ) 5 nM of PTX and ( H – J ) 86 nM of DOX or with solvent (Ctrl). ( B , E , H ) Cell viability percentage was determined by ATP level measurement and results were expressed as a percentage of the control exposed to the solvent. ( C , F , I ) HCC1806 cells were co-labeled with annexin V coupled to FITC and with propidium iodide and the living, early- and late-apoptotic cells were analyzed by flow cytometry. ( D , G , J ) Apoptosis was also analyzed by Western blot by studying the cleavage of PARP-1 (cPARP) and caspase 7 (cCASP7) markers. Relative protein level expressions correspond to the protein of interest on GAPDH ratios. The values represent the means ± SEM of four to eight different experiments. A p -value of less than 0.05 was considered statistically significant with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The uncropped blots are shown in .

Article Snippet: The anti-cleaved caspase 7 antibody (#9494S, Cell Signaling Technology, Saint-Cyr-L’École, France) was diluted at 1:800, the anti-cleaved PARP-1 antibodies (552596, BD Biosciences, Le Pont de Claix, France) and anti-CLDN1 (C5838-02C, US Biological, Nanterre, France) were diluted at 1:1000 and the GAPDH antibody (60004-1-IgG, ProteinTech, Illkirch-Graffenstaden, France) was diluted at 1:5000.

Techniques: Transfection, Expressing, Western Blot, Labeling, Flow Cytometry

Journal: Cancers

Article Title: CLDN1 Sensitizes Triple-Negative Breast Cancer Cells to Chemotherapy

doi: 10.3390/cancers14205026

Figure Lengend Snippet: CLDN1 expression is involved in MDA-MB-231 cells sensitivity to PTX and DOX. MDA-MB-231 cells were transfected with a siRNA directed against claudin-1 (siCLDN1) or with a scramble scRNA. 7 h after transfection, cells were treated for 72 h with ( A – C ) 5 nM of PTX, ( D – F ) 86 nM of DOX or with solvent (Ctrl). ( A , D ) CLDN1 expression was studied by western blot. ( B , E ) MDA-MB-231 cells were co-labeled with annexin V coupled to FITC and with propidium iodide and the living, early- and late-apoptotic cells were analyzed by flow cytometry. ( C , F ) Cleavage of apoptotic markers PARP-1(cPARP) and caspase 7 (cCASP7) expressions were analyzed by Western blot. Relative protein level expressions correspond to the protein of interest on GAPDH ratios. The values represent the means ± SEM of three to five different experiments. A p -value of less than 0.05 was considered statistically significant with * p < 0.05, ** p < 0.01. The uncropped blots are shown in .

Article Snippet: The anti-cleaved caspase 7 antibody (#9494S, Cell Signaling Technology, Saint-Cyr-L’École, France) was diluted at 1:800, the anti-cleaved PARP-1 antibodies (552596, BD Biosciences, Le Pont de Claix, France) and anti-CLDN1 (C5838-02C, US Biological, Nanterre, France) were diluted at 1:1000 and the GAPDH antibody (60004-1-IgG, ProteinTech, Illkirch-Graffenstaden, France) was diluted at 1:5000.

Techniques: Expressing, Transfection, Western Blot, Labeling, Flow Cytometry

Journal: Cancers

Article Title: CLDN1 Sensitizes Triple-Negative Breast Cancer Cells to Chemotherapy

doi: 10.3390/cancers14205026

Figure Lengend Snippet: CLDN1 overexpression sensitizes Hs578T cells to PTX and DOX but not to 5-FU. ( A ) Hs578T cells stably transfected with CLDN1 vector (Hs578T/CLDN1) or the empty vector (Hs578T/CTRL) were treated for 72 h with ( B , C ) PTX at 5 nM, ( D , E ) DOX at 210 nM and ( F , G ) 5-FU at 181 µM, or with solvent (Ctrl). ( A ) CLDN1 expression was studied by western blot. ( B , D , F ) Hs578T cells were co-labeled with annexin V coupled to FITC and with propidium iodide and the living, early- and late-apoptotic cells were analyzed by flow cytometry. ( C , E , G ) Cleavage of apoptotic markers PARP-1 (cPARP) and caspase 7 (cCASP7) expression were analyzed by Western blot. Relative protein level expressions correspond to the protein of interest on GAPDH ratios. The values represent the means ± SEM of three to four different experiments. A p -value of less than 0.05 was considered statistically significant with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The uncropped blots are shown in .

Article Snippet: The anti-cleaved caspase 7 antibody (#9494S, Cell Signaling Technology, Saint-Cyr-L’École, France) was diluted at 1:800, the anti-cleaved PARP-1 antibodies (552596, BD Biosciences, Le Pont de Claix, France) and anti-CLDN1 (C5838-02C, US Biological, Nanterre, France) were diluted at 1:1000 and the GAPDH antibody (60004-1-IgG, ProteinTech, Illkirch-Graffenstaden, France) was diluted at 1:5000.

Techniques: Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Western Blot, Labeling, Flow Cytometry

Journal: Cancers

Article Title: CLDN1 Sensitizes Triple-Negative Breast Cancer Cells to Chemotherapy

doi: 10.3390/cancers14205026

Figure Lengend Snippet: CLDN1 expression sensitizes MDA-MB-231 cells to 5-FU. MDA-MB-231 cells stably transfected with CLDN1 vector (MDA-MB-231/CLDN1) or the empty vector (MDA-MB-231/CTRL) were treated for 72 h with 5-FU at 50 µM or with solvent (Ctrl). ( A ) CLDN1 expression was studied by western blot. ( B ) Cells were co-labeled with annexin V coupled to FITC and with propidium iodide and the living, early- and late-apoptotic cells were analyzed by flow cytometry. ( C ) Cleavage of apoptotic markers PARP-1 (cPARP) and caspase 7 (cCASP7) expressions were analyzed by Western blot. Relative protein level expressions correspond to the protein of interest on GAPDH ratios. The Western blot picture compared MDA-MB-231/CLDN1 non-treated cells (Ctrl) and 5 FU treated cells. The values represent the means ± SEM of three to four different experiments. A p -value of less than 0.05 was considered statistically significant with * p < 0.05, ** p < 0.01, *** p < 0.001. The uncropped blots are shown in .

Article Snippet: The anti-cleaved caspase 7 antibody (#9494S, Cell Signaling Technology, Saint-Cyr-L’École, France) was diluted at 1:800, the anti-cleaved PARP-1 antibodies (552596, BD Biosciences, Le Pont de Claix, France) and anti-CLDN1 (C5838-02C, US Biological, Nanterre, France) were diluted at 1:1000 and the GAPDH antibody (60004-1-IgG, ProteinTech, Illkirch-Graffenstaden, France) was diluted at 1:5000.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Labeling, Flow Cytometry

Journal: Cell Reports

Article Title: Stromal Cell-Contact Dependent PI3K and APRIL Induced NF-κB Signaling Prevent Mitochondrial- and ER Stress Induced Death of Memory Plasma Cells

doi: 10.1016/j.celrep.2020.107982

Figure Lengend Snippet: Contact with Stromal Cells Inhibits Activation of Caspase 3 and 7, whereas APRIL Inhibits Activation of Caspase 12 Expression of activated caspase 3 (A), caspase 7 (B), and caspase 12 (C) of CD138 + PCs determined by flow cytometry, shown as geometric mean fluorescence intensity, on day 1 in PCs cultured under the indicated conditions (pooled from a minimum of two independent experiments with n = 6–12 technical replicates for each group). Statistics: Kruskal-Wallis test (activated Casp3), ordinary one-way ANOVA (activated Casp7/Casp12).

Article Snippet: Anti-mouse active caspase 7 , Cell Signaling Technology , Catalog # 8438T; RRID: AB_11178377.

Techniques: Activation Assay, Expressing, Flow Cytometry, Fluorescence, Cell Culture

Journal: Cell Reports

Article Title: Stromal Cell-Contact Dependent PI3K and APRIL Induced NF-κB Signaling Prevent Mitochondrial- and ER Stress Induced Death of Memory Plasma Cells

doi: 10.1016/j.celrep.2020.107982

Figure Lengend Snippet:

Article Snippet: Anti-mouse active caspase 7 , Cell Signaling Technology , Catalog # 8438T; RRID: AB_11178377.

Techniques: Recombinant, Electron Microscopy, Staining, Software

Journal: Molecular Therapy

Article Title: Activation of Nrf2 Signaling Augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven Suppression of Antiviral Immunity

doi: 10.1016/j.ymthe.2017.04.022

Figure Lengend Snippet: Sulforaphane Enhances VSVΔ51-Mediated Oncolytic Activity in Resistant PC-3 Cells PC-3 cells were pretreated for 24 hr with increasing concentrations of sulforaphane (SFN) and were subsequently infected with VSVΔ51-GFP (MOI 1). (A and B) Infectivity was determined by fluorescent microscopy (A) or quantified by flow cytometry (B) at the indicated times. (C) Viral replication was assessed by plaque assay. (D) Cell survival was imaged by light microscopy 72 hr following VSVΔ51 challenge. (E and F) The percentage of viable (E) and apoptotic cells (F) was assessed using an annexin-V/7AAD staining by flow cytometry at the indicated times. (A–F) Data are the means ± SEM from two experiments performed in triplicate. (G) The correlation between the percentage of VSVΔ51-GFP + cells at 24 hr and the percentage of apoptotic annexin-V + cells at 48 hr was calculated in PC-3 cells using a Spearman test (n = 33). (H) The same statistical test was used to calculate the correlation between the percentage of apoptotic annexin-V + cells and the percentage of MitoSOX + cells at 48 hr after infection (n = 36). (I) PC-3 cells were pretreated for 24 hr with SFN (20 μM) and challenged with VSVΔ51-GFP (MOI 1) for 48 hr. Whole-cell extracts (WCEs) were analyzed by immunoblotting for cleaved caspase-3, cleaved caspase-7 (high and low exposure), cleaved PARP, VSVΔ51-GFP, and β-actin. Results are from a representative experiment; all immunoblots are from the same samples.

Article Snippet: Blots were blocked for 1 hr with 5% nonfat dried milk in PBST (PBS + 0.1% Tween 20) and then probed overnight at 4°C with any of the following specific primary antibodies: VSV-whole virus antisera, anti-GFP (sc-9996, Santa Cruz), anticleaved caspase-3 (9661, Cell Signal), anti-caspase-7/cleaved caspase-7 (9494, Cell Signal), anti-PARP/cleaved PARP (9541, Cell Signal), anti-Nrf2 (ab62352, Abcam; 12721, Cell Signal), anti-HO-1 (5853, Cell Signal), anti-P-IRF3 (4947, Cell Signal), anti-IRF3 (ab68481, Abcam), anti-P-STAT1 (9167, Cell Signal), anti-STAT1 (sc-417, Santa Cruz), anti-RIG-I (Bleed, Millipore), anti-STING (13647, Cell Signal), anti-LC3B (2775, Cell Signal), anti-Atg7 (8558, Cell Signal), and anti-actin (MAB1501, Millipore) used as loading control.

Techniques: Activity Assay, Infection, Microscopy, Flow Cytometry, Plaque Assay, Light Microscopy, Staining, Western Blot