Journal: Oncotarget

Article Title: Identification of breast cancer cell subtypes sensitive to ATG4B inhibition

doi: 10.18632/oncotarget.11408

Figure Lengend Snippet: A. HER2-positive cell lines have higher protein levels of ATG4B as compared to HER2-negative cell lines. Representative western blot analysis shows ATG4B basal expression in a panel of HER2-positive (n=5) and HER2-negative (n=5) breast cancer cell lines. Bar plots demonstrate average ATG4B expression within each group of cell lines (mean±SEM) normalized to actin (used as internal control for protein loading); n=3; P values are based on the Student's t -test. B. HER2+ overexpression in MCF7 and MDA-MB-231-BR-eGFP breast cancer cells results in elevated ATG4B protein levels. Representative western blot and bar plots show ATG4B expression normalized to actin loading control (mean±SEM; n=3); P values are based on the Student's t -test. C. HER2 knockdown in three HER2-overexpressing cell lines (SKBR3, MDA-MB-453, and JIMT1) results in a decrease in ATG4B protein levels, increase in LC3B-II and decrease in SQSTM1/p62 expression. Representative western blot from 3 independent experiments. Bar graph on right shows ATG4B expression normalized to actin loading control; (mean±SEM; n=3); P values are based on the one-way ANOVA with Dunnett post-test.

Article Snippet: SKBR3 cells (ATCC) were maintained in McCoy's 5A (modified) medium with 10% FBS.

Techniques: Western Blot, Expressing, Control, Over Expression, Knockdown

Journal: Oncotarget

Article Title: Identification of breast cancer cell subtypes sensitive to ATG4B inhibition

doi: 10.18632/oncotarget.11408

Figure Lengend Snippet: A. HER2-overexpressing cells have decreased basal autophagy levels. Western blot flux assay using saturating (60nM) concentrations of Bafilomycin A1 (Baf A1) shows LC3B-II and p62 levels in parental MCF7 cells as compared to HER2-overexpressing MCF7 cells. Bar graphs on right show LC3B-II (in the presence of Baf A1) and p62 expression, normalized to actin loading control (fold change); mean±SEM; n=3; P values are based on Student's t-test. B. HER2 knockdown increases autophagic flux. SKBR3 cells stably expressing mRFP-EGFP-LC3B protein were treated with either HER2 or scramble siRNA under fed and starved conditions. Increase in red puncta (autolysosomes) relative to yellow puncta (autophagosomes) indicates increased autophagic flux in response to HER2 knockdown. Bar graphs show average (mean±SEM) ratio of red to yellow puncta per cell. Data was collected from 3 independent experiments, and P values are based on the one-way ANOVA with Dunnett post-test. Scale bar, 50 μm. C. HER2 knockdown increases autophagic flux. Western blot flux assay using saturating (60nM) concentrations of Bafilomycin A1 (Baf A1) shows LC3B-II, p62, and ATG4B levels in cells treated with HER2 siRNAs as compared to scramble siRNA control. Bar graphs on right show LC3B-II (in the presence of Baf A1), p62, and ATG4B expression, normalized to actin loading control (fold change); mean±SEM; n=3; P values are based on Student's t-test.

Article Snippet: SKBR3 cells (ATCC) were maintained in McCoy's 5A (modified) medium with 10% FBS.

Techniques: Western Blot, Flux Assay, Expressing, Control, Knockdown, Stable Transfection

Journal: Oncotarget

Article Title: Identification of breast cancer cell subtypes sensitive to ATG4B inhibition

doi: 10.18632/oncotarget.11408

Figure Lengend Snippet: A. ATG4B knockdown results in inhibition of autophagy. Western blot flux assay using saturating concentrations of Bafilomycin A1 (Baf A1) show LC3B-II and p62 levels in HER2+MCF7 cells following ATG4B knockdown under fed and starved conditions. Bar graphs on right show LC3B-II (in the presence of Baf A1) and p62 expression, normalized to actin loading control (fold change); mean±SEM; n=3; P values are based on Student's t-test. B. ATG4B knockdown prevents HER2 positive cells from increasing autophagic flux in response to starvation. SKBR3 cells stably expressing mRFP-EGFP-LC3B protein were treated with either ATG4B or scramble siRNA under fed and starved conditions. Bar graphs show average (mean±SEM) ratio of red to yellow puncta per cell. Data was calculated from 3 independent experiments, and P values are based on the one-way ANOVA with Dunnett post-test. Scale bar, 50 μm. C. HER2-overexpressing cells are sensitive to ATG4B inhibition under starvation. HER2-negative (MDA-MB-231, SUM159PT, Hs578T, and MCF7) and HER2-positive (SKBR3, MDA-MB-453, JIMT-1, and HER2+MCF7) cells were treated twice with ATG4B siRNA under starved conditions, and colonies were visualized with crystal violet staining. Retained crystal violet staining was measured by absorbance at 590nm (A590) to generate a proliferation index. Bar plot indicates mean±SEM values from 3 independent experiments normalized to fed scramble controls. P values are based on the one-way ANOVA with Dunnett post-test. D. ATG4B knockdown under starvation results in caspase-dependent cell death in HER2-overexpressing cells. SKBR3, MDA-MB-453, JIMT-1, and HER2+MCF7 cells were assayed for induction of caspase-3/7 activity using the luminescence (RLU, relative luminescence unit; y axis)-based Caspase-Glo assay. Bar plots show mean±SEM values from 3 independent experiments. Cycloheximide (CHX) was used as a positive control for caspase activation. P values are based on the one-way ANOVA with Dunnett post-test.

Article Snippet: SKBR3 cells (ATCC) were maintained in McCoy's 5A (modified) medium with 10% FBS.

Techniques: Knockdown, Inhibition, Western Blot, Flux Assay, Expressing, Control, Stable Transfection, Staining, Activity Assay, Caspase-Glo Assay, Positive Control, Activation Assay

Journal: Oncotarget

Article Title: Identification of breast cancer cell subtypes sensitive to ATG4B inhibition

doi: 10.18632/oncotarget.11408

Figure Lengend Snippet: A. Combination ATG4B and HER2 knockdown results in autophagy flux inhibition in HER2-positive cells. SKBR3 cells stably expressing mRFP-EGFP-LC3B protein were treated with HER2 and ATG4B siRNA (ATG4B siRNA1, center; ATG4B siRNA2, right) or scramble siRNA (control, left) under fed and starved conditions. Bar graphs show average (mean±SEM) ratio of red to yellow puncta per cell. Data shown is from 3 independent experiments, and P values are based on the one-way ANOVA with Dunnett post-test. Scale bar, 50 μm. B. HER2 knockdown results in decreased ATG4B levels, whereas ATG4B and ATG4B+HER2 knockdowns lead to a further decrease in ATG4B levels. Representative western blot analysis shows ATG4B levels in SKBR3-tfLC3B cells following ATG4B and HER2 knockdowns alone or combined. C. HER2 knockdown results in decreased ATG4A, ATG4C and ATG4D levels. Representative (n=2) western blot analysis shows ATG4A, ATG4C, and ATG4D levels in SKBR3-tfLC3B cells following HER2 knockdown. D. ATG4C (under both fed and starved conditions) and ATG4A (under starvation only) are required for autophagy induction following HER2 knockdown. SKBR3 cells stably expressing mRFP-EGFP-LC3B protein were treated with ATG4A, ATG4C, ATG4D or scramble siRNA alone or in combination with HER2 siRNA under fed and starved conditions. Bar graphs show average (mean±SEM) ratio of red to yellow puncta per cell. Data shown is from 3 independent experiments, and P values are based on the one-way ANOVA with Dunnett post-test. Scale bar, 10 μm.

Article Snippet: SKBR3 cells (ATCC) were maintained in McCoy's 5A (modified) medium with 10% FBS.

Techniques: Knockdown, Inhibition, Stable Transfection, Expressing, Control, Western Blot

Journal: Oncotarget

Article Title: Identification of breast cancer cell subtypes sensitive to ATG4B inhibition

doi: 10.18632/oncotarget.11408

Figure Lengend Snippet: A. and B. ATG4B is required for cell survival under HER2 inhibition. Combination ATG4B and HER2 knockdown results in decreased cell viability as compared to single knockdowns under fed and starved conditions. Alamar blue (A) and Trypan Blue exclusion (B) assays were used to assess cell viability following siRNA treatment. Bar graphs represent mean±SEM values of %viable cells relative to fed scramble controls (n=3 independent experiments). P values are based on the one-way ANOVA with Dunnett post-test. C. Trastuzumab (Tz) treatment results in increased autophagic flux and decreased levels of ATG4B. Representative western blots for SKBR3-tfLC3, MDA-MB-453, and JIMT-1 cells. Bar graphs on right show LC3B-II (in the presence of Baf A1), p62, and ATG4B expression, normalized to actin loading control (fold change); mean±SEM; n=3; P values are based on Student's t-test. D. ATG4B knockdown sensitizes HER2-positive cells to trastuzumab (Tz). SKBR3, MDA-MB-453, and JIMT-1 cells were pre-treated with ATG4B and scramble siRNAs, and then treated with trastuzumab for 72 hours. Colonies were visualized with crystal violet staining (on the left). Retained crystal violet staining was measured by A590 to generate a proliferation index (central panel). Bar plot indicates mean±SEM values from 3 independent experiments normalized to scramble siRNA controls. P values are based on the one-way ANOVA with Dunnett post-test. Accompanying representative western blot analysis of ATG4B levels, as well as flux assay of LC3B-II levels using saturating concentrations of Bafilomycin A1 (Baf A1) is shown on the right.

Article Snippet: SKBR3 cells (ATCC) were maintained in McCoy's 5A (modified) medium with 10% FBS.

Techniques: Inhibition, Knockdown, Western Blot, Expressing, Control, Staining, Flux Assay

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: A. HCT116 cells were cultured in the presence of 1 µM b-AP15 or 100 nM bortezomib and harvested at the indicated time points. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, HSP-70B', p21, PARP or β-actin (loading control). B. HCT116 cells or hTERT-RPE1 cells were exposed to different concentrations of b-AP15 for 1 hour, followed by washing and incubation for 16 hours in drug-free medium. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, PARP and β-actin (loading control).

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: Cell Culture, Western Blot, Control, Incubation

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: A. and B. HCT116 cells were treated for 1 h with DMSO, tunicamycin (10 µg/ml) or CAM741 (10 µM) before pulse-labelling for 10 min with [ 35 S] Met/Cys. Endogenous prosaposin (pSAP) was recovered by immunoprecipitation and newly synthesised pSAP species were visualised by phosphorimaging. In DMSO-treated cells, pSAP was fully glycosylated (pSAP-5), whereas Endo H digestion or tunicamycin treatment yielded non-glycosylated pSAP (pSAP-0). Inhibition of protein translocation into the ER by CAM741 resulted in the appearance of a pSAP species that migrated more slowly than the non-glycosylated protein and was Endo H-resistant. This species may represent signal sequence-containing preprosaposin (prepSAP-0) that has failed to translocate across the ER membrane. C. Distinct forms of endogenous pSAP in HCT116 cells treated with DMSO, b-AP15 (1 µM), bortezomib (20 nM), cpdA (10 µM) or tunicamycin (10 µg/ml) were recovered by immunoprecipitation and visualised by phosphorimaging. Treatment with cpdA specifically inhibits the co-translational translocation of pSAP into the ER as judged by the appearance of prepSAP-0 species.

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: Immunoprecipitation, Inhibition, Translocation Assay, Sequencing, Membrane

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: A. HCT116 cells were pre-treated with or without (control) 10 µM cpdA for 16 hours, exposed to the indicated concentration of b-AP15 for 1 hour, then incubated for a further 16 hours in drug-free medium. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, PARP and β-actin (loading control). B. HCT116 cells were treated as above. Following treatment, the number of dead cells was measured by Trypan-blue staining. Bar chart shows mean +/− SD of three independent experiments. Statistical significance was calculated using the Student's t-test. P values * = 0.05 and ** = 0.01. C. and D. hTERT-RPE1 cells were treated and analyzed as above. E. and F. Human diploid fibroblasts were treated and analyzed as above, and the number of dead cells was determined 36 hours after drug treatment.

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: Control, Concentration Assay, Incubation, Western Blot, Staining

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: A. HCT116 cells were exposed to 1 µM b-AP15 or 100 nM bortezomib (BZ) for 8 hours or 16 hours, in the presence or absence of 1 mM cysteine as indicated. Cell lysates were subjected to immunoblotting for LC3-I/II and β-actin (loading control). B. HCT116 cells were exposed to 1 µM b-AP15 for 16 hours in the presence or absence of 1 mM cysteine. Where indicated, cells were pre-treated with 10 µM cpdA for 16 hours prior to b-AP15 treatment. Apoptosis was determined by the appearance of caspase-cleaved K18. Bar chart shows mean values ± SD of of three independent experiments.

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Elevation of Proteasomal Substrate Levels Sensitizes Cells to Apoptosis Induced by Inhibition of Proteasomal Deubiquitinases

doi: 10.1371/journal.pone.0108839

Figure Lengend Snippet: Treatment of HCT116 cells with b-AP15 and cpdA does not deplete cellular cysteine levels.

Article Snippet: HCT116 colon carcinoma cells (ATCC) were maintained in McCoy's 5A modified medium/10% fetal calf serum, hTERT-RPE1 cells in DMEM:F12 medium/10% fetal calf serum, human diploid fibroblasts and HeLa cells (European Collection of Cell Cultures) were maintained in DMEM medium/10% fetal calf serum and MelJuSo-Ub G76V -YFP cells in Iscove's DMEM medium/10% fetal calf serum.

Techniques: