Journal: Oncotarget

Article Title: CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways

doi:

Figure Lengend Snippet: A. PK45-p and B. PK59 cells were treated with CGK733 for 6 h before being processed for Western blot analysis using antibodies against LC3 A/B and actin. C. PK59 cells were treated with CGK733 for 6 h before being processed for immunofluorescence using an antibody against LC3 A/B. Quantification of the samples are expressed as the number of endogenous LC3 puncta per cell. D. PK45-p cells were transfected with TagGFP2-LC3 Lentivirus for 48 h and then treated with CGK733 for 6 h before observation by fluorescent microscopy. Quantification of the samples are expressed as the number of GFP-LC3 puncta per cell. Error bars represent the standard deviations from counting 30 cells in three independent experiments. ***, p < 0.001.

Article Snippet: CGK733 (sc-202964), 3-MA (sc-205596) and Compound C (sc-200689) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Immunofluorescence, Transfection, Microscopy

Journal: Oncotarget

Article Title: CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways

doi:

Figure Lengend Snippet: A. PK59 cells were treated with CGK733 for 6 h in the presence or absence of 10 μM of CQ before analysis by immunofluorescence using an antibody against LC3 A/B. The lower figure indicates the related quantification of the endogenous LC3 puncta per cell. Error bars represent the standard deviations from counting 30 cells in three independent experiments. **, p < 0.01. B. PK45- p cells were treated with CGK733 for 6 h in the presence or absence of 10 μM of CQ before analysis by Western blot. C. PK59 cells were treated with CGK733 or rapamycin for 6 h in the presence or absence of 10 μM of CQ before analysis by Western blot. D. PK45- p cells were treated with CGK733 for 6 h before performing immunoprecipitation using an antibody against LC3 A/B. 1% of the original samples were used as inputs. E. PK45- p cells were transfected with TagGFP2-LC3 Lentivirus for 48 h and then treated with CGK733 before analysis by immunofluorescence using an antibody against p62. CQ was added in this experiment in order to easily observe autophagosomes in non-treated cells.

Article Snippet: CGK733 (sc-202964), 3-MA (sc-205596) and Compound C (sc-200689) were purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Western Blot, Immunoprecipitation, Transfection

Journal: Oncotarget

Article Title: CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways

doi:

Figure Lengend Snippet: A. PK45- p and PK59 cells were treated with CGK733 for 6 h before being processed for Western blot analysis. B. PK45- p cells were treated with CGK733 for 6 h after transfection with AMPK siRNA for 48 h. C. PK45- p cells were treated with CGK733 for 6 h in the presence or absence of 1 μM of PERK inhibitor (GSK2606414) before analysis by Western blot. D. PK45- p cells were treated with CGK733 for 6 h after transfection with CHOP siRNA for 48 h. E. PK45- p cells were treated with CGK733 for 6 h after transfection with AMPK and/or CHOP siRNAs for 48 h.

Article Snippet: CGK733 (sc-202964), 3-MA (sc-205596) and Compound C (sc-200689) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Transfection

Journal: Oncotarget

Article Title: CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21 Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways

doi:

Figure Lengend Snippet: A. PK45- p cells were treated with CGK733 for 6 h after transfection with LC3 B siRNA for 48 h. B. PK45- p cells were treated with CGK733 for 6 h after transfection with EGFP-LC3 B plasmid for 48 h. C. PK45- p cells were treated with CGK733 for 6 h after the transfection with LC3 A siRNA for 48 h. D. The scheme indicates that CGK733 induced the expression of p21 Waf1/Cip1 through the LC3 B-mediated AMPK and PERK/CHOP activation.

Article Snippet: CGK733 (sc-202964), 3-MA (sc-205596) and Compound C (sc-200689) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Expressing, Activation Assay

Journal: JCI Insight

Article Title: AURKA inhibitor VIC-1911 induces mitotic defects and functional BRCAness, sensitizing prostate cancer to PARP inhibition

doi: 10.1172/jci.insight.196665

Figure Lengend Snippet: ( A ) Immunoblotting analysis shows a dose-dependent increase in cleaved caspase-3 and phospho-γH2AX (S139) in response to 48 hours of incubation with increasing doses of VIC-1911. ( B ) VIC-1911 treatment induces mitotic abnormalities. Representative immunofluorescent images show increased nuclear-specific phospho-γH2AX (S139) staining in C4-2B cells with mitotic defects after 24 hours of treatment with 0.1 μM of VIC-1911. Scale bar: 30 μm. ( C ) Immunoblotting images show a gradually increasing level of phospho-ATM (S1981) in pKAP1 (S824) in response to 24 hours of treatment with increasing doses of VIC-1911. ( D ) Immunoblotting images show a gradually increasing level of phospho-ATR (T1989) in response to 24 hours of treatment with increasing doses of VIC-1911. ( E ) VIC-1911 in combination with lartesertib (ATMi) reduces the proliferation of AR-positive CRPC cells. Relative cell confluence was assessed using IncuCyte live-cell imaging. ( F ) VIC-1911 in combination with ceralasertib (ATRi) reduces the proliferation of AR-positive CRPC cells. Relativ e cell confluence was assessed using IncuCyte live-cell imaging. Bliss coefficient (S): S = 0 indicates an additive effect, S > 0 indicates synergy, and S < 0 indicates antagonism. The IncuCyte experiments were run once.

Article Snippet: Olaparib, talazoparib, and nocodazole were purchased from TargetMol, and ceralasertib (ATRi) and lartesertib (ATMi) were purchased from MedChemExpress.

Techniques: Western Blot, Incubation, Staining, Live Cell Imaging

Journal: Biomolecules

Article Title: ATM Inhibitor Suppresses Gemcitabine-Resistant BTC Growth in a Polymerase θ Deficiency-Dependent Manner

doi: 10.3390/biom10111529

Figure Lengend Snippet: Knockdown of DNA polymerase θ increases AZD0156 sensitivity in BTC cells. ( A ) Cell viability in various concentrations of AZD0156. The values (means ± SD) are from two data points and expressed as a percentage relative to that of the cells without AZD0156 treatment. IC 50 values for AZD0156 in the BTC cell lines are shown in each panel. ( B – D ) DNA polymerase θ was depleted by shRNAs (shPOLQ) in SSP-25 cells ( B ), TFK-1 cells, ( C ) or TGBC-24TKB cells ( D ). Left: the relative mRNA level of POLQ. The values (means ± SD) are from two independent experiments, and are presented as fold-change relative to the level of the cells receiving shRNAs against LacZ (shLacZ). * p < 0.05 by Student’s t -test. Right: the cell viability in various concentrations of AZD0156. The values (means ± SD) are from two data points, and are expressed as a percentage relative to that of the cells without AZD0156 treatment. The IC 50 values for AZD0156 in the cell lines are shown in the panel. ( E ) Cell proliferation assay. A total of 5 × 10 3 cells were seeded in a 96-well plate for 16–24 h, and were then cultured in the absence or presence of 1.25 μM AZD0156 for 0, 24, 48, or 72 h. Cell viability was quantified by CCK-8. The values (means ± SD) are from two independent experiments, and are presented as the fold change relative to the baseline (0 h). * p < 0.05 by Student’s t -test. ( F – H ) Cell proliferation assay. DNA polymerase θ was depleted by shRNAs (shPOLQ) in SSP-25 cells ( F ), TFK-1 cells ( G ), or TGBC-24TKB cells ( H ). A total of 5 × 10 3 cells were seeded in a 96-well plate for 16–24 h, and were then cultured in the absence or presence of 1.25 μM (F), 5 μM (G), or 1 μM (H) AZD0156 for 0, 24, 48, or 72 h. The cell viability was quantified by CCK-8. The values (means ± SD) are from two independent experiments, and are presented as the fold change relative to the baseline (0 h). * p < 0.05 by Student’s t -test.

Article Snippet: AZD0156 (ATM inhibitor), gemcitabine, and cisplatin were purchased from AdooQ BioScience (Irvine, CA, USA).

Techniques: Knockdown, Proliferation Assay, Cell Culture, CCK-8 Assay

Journal: Biomolecules

Article Title: ATM Inhibitor Suppresses Gemcitabine-Resistant BTC Growth in a Polymerase θ Deficiency-Dependent Manner

doi: 10.3390/biom10111529

Figure Lengend Snippet: DNA damage enhances the effect of AZD0156-mediated cell death in DNA polymerase θ-depleted BTC cells. ( A ) Cell viability in various concentrations of AZD0156. TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ) or LacZ (shLacZ) were treated with 5 Gy photons, and 1 μM H2O2 for 2 h before AZD0156 addition. The values (means ± SD) are from two data points, and are expressed as a percentage relative to that of the cells without AZD0156 treatment. The IC 50 values for AZD0156 in the cell lines are shown in each panel. ( B ) The relative cell viability in 0.5 μM AZD0156 for 72 h. TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ+) or LacZ (shPOLQ–) were treated with 5 Gy photons and 1 μM H 2 O 2 for 2 h before AZD0156 addition. The values (means ± SD) are from three independent experiments, and are expressed as a percentage relative to that of the cells without AZD0156, 5 Gy photons, and 1 μM H 2 O 2 treatment; * p < 0.05 by Student’s t -test. ( C ) Cell viability in various concentrations of AZD0156. TFK-1 cells receiving shRNAs specific to DNA polymerase θ (shPOLQ) or LacZ (shLacZ) were treated with 5 Gy photons and 1 μM H 2 O 2 for 2 h before AZD0156 addition. The values (means ± SD) are from two data points, and are expressed as a percentage relative to that of the cells without AZD0156 treatment. The IC 50 values for AZD0156 in the cell lines are shown in each panel. ( D ) The relative cell viability in 2.5 μM AZD0156 for 72 h. TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ+) or LacZ (shPOLQ–) were treated with 5 Gy photons and 1 μM H 2 O 2 for 2 h before AZD0156 addition. The values (means ± SD) are from two independent experiments, and are expressed as a percentage relative to that of the cells without AZD0156, 5 Gy photons, and 1 μM H 2 O 2 treatment; * p < 0.05 by Student’s t -test. ( E ) Western blots showing the level of H2AX Ser139 phosphorylation (γ-H2AX), PARP1, ATM Ser1981 phosphorylation, and total ATM in TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ) or LacZ (shLacZ). The cells were treated with 5 Gy photons and 1 μM H 2 O 2 (+) or control H 2 O (–) for 2 h, and then cultured in the absence (–) or presence (+) of 2.5 μM AZD0156 for another 4 h; α-tubulin was the loading control. ( F , G ) Micronucleus frequency in TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ) or LacZ (shLacZ). The cells were treated with 5 Gy photons and 1 μM H 2 O 2 (+) or control H 2 O (–) for 2 h, and were then cultured in the absence (–) or presence (+) of 2.5 μM AZD0156 for another 24 h. Representative images of the micronucleus are (arrows) are shown in panel F. The quantitative data are shown in panel G. The values (means ± SD) are from two independent experiments; * p < 0.05 by Student’s t -test.

Article Snippet: AZD0156 (ATM inhibitor), gemcitabine, and cisplatin were purchased from AdooQ BioScience (Irvine, CA, USA).

Techniques: Western Blot, Phospho-proteomics, Control, Cell Culture

Journal: Biomolecules

Article Title: ATM Inhibitor Suppresses Gemcitabine-Resistant BTC Growth in a Polymerase θ Deficiency-Dependent Manner

doi: 10.3390/biom10111529

Figure Lengend Snippet: Knockdown of DNA polymerase θ improves AZD0156-mediated cell death. ( A ) DNA polymerase θ was depleted by shRNAs (shPOLQ; #1 and #2) in TGBC-24TKB cells. The relative mRNA level of POLQ is shown. The values (means ± SD) are from two independent experiments, and are presented as the fold change relative to the level of the cells receiving shRNAs against LacZ (shLacZ); * p < 0.05 by Student’s t -test. ( B ) The cell viability in various concentrations of AZD0156. TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ; #1 and #2) or LacZ (shLacZ) were treated with 0.5 μM GEM and 5 μM CDDP for 24 h before AZD0156 addition. The values (means ± SD) are from two data points, and are expressed as a percentage relative to that of the cells without AZD0156 treatment. The IC50 values for AZD0156 in the cell lines are shown in each panel. ( C ) The relative cell viability in 0.5 μM AZD0156 for 72 h. TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ; #1 and #2) or LacZ (shLacZ) were treated with 0.5 μM GEM and 5 μM CDDP for 24 h before AZD0156 addition. The values (means ± SD) are from three independent experiments, and are expressed as a percentage relative to that of the cells receiving shRNAs against LacZ; * p < 0.05 by Student’s t -test. ( D ) DNA polymerase θ was depleted by shRNAs (shPOLQ; #1 and #2) in TFK-1 cells. The relative mRNA level of POLQ. The values (means ± SD) are from two independent experiments, and are presented as the fold change relative to the level of the cells receiving shRNAs against LacZ (shLacZ); * p < 0.05 by Student’s t -test. ( E ) The cell viability in various concentrations of AZD0156. TFK-1 cells receiving shRNAs specific to DNA polymerase θ (shPOLQ; #1 and #2) or LacZ (shLacZ) were treated with 1 μM GEM and 10 μM CDDP for 24 h before AZD0156 addition. The values (means ± SD) are from two data points, and are expressed as a percentage relative to that of the cells without AZD0156 treatment. The IC 50 values for AZD0156 in the cell lines are shown in each panel. ( F ) The relative cell viability in 2.5 μM AZD0156 for 72 h. TFK-1 cells receiving shRNAs specific to DNA polymerase θ (shPOLQ; #1 and #2) or LacZ (shLacZ) were treated with 1 μM GEM and 10 μM CDDP for 24 h before AZD0156 addition. The values (means ± SD) are from three independent experiments, and are expressed as a percentage relative to that of the cells receiving shRNAs against LacZ. * p < 0.05 by Student’s t -test. ( G ) Western blots showing the level of H2AX Ser139 phosphorylation (γ-H2AX), PARP1, ATM Ser1981 phosphorylation, and total ATM in TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ; #1 and #2) or LacZ (shLacZ). The cells were treated with 0.5 μM GEM and 5 μM CDDP for 24 h, and were then cultured in the absence (–) or presence (+) of 2.5 μM AZD0156 for another 4 h; α-tubulin was the loading control. ( H ) The cells described in panel G were fixed and stained with H2AX Ser139 phosphorylation (γ-H2AX) and nuclei. Scale bar = 10 µm. ( I ) The relative fluorescent intensity of γ-H2AX was measured for each field ( n = 5) and expressed as the fold change relative to the level of the cells without chemotherapy drugs and AZD0156 treatment. The values (means ± SD) are from three independent experiments; * p < 0.05 by Student’s t -test.

Article Snippet: AZD0156 (ATM inhibitor), gemcitabine, and cisplatin were purchased from AdooQ BioScience (Irvine, CA, USA).

Techniques: Knockdown, Western Blot, Phospho-proteomics, Cell Culture, Control, Staining

Journal: Biomolecules

Article Title: ATM Inhibitor Suppresses Gemcitabine-Resistant BTC Growth in a Polymerase θ Deficiency-Dependent Manner

doi: 10.3390/biom10111529

Figure Lengend Snippet: AZD0156 and DNA polymerase θ depletion leads to DNA damage accumulation and suppresses tumor growth. ( A ) The alkaline comet assay: TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ) or LacZ (shLacZ) were treated with 0.5 μM GEM and 5 μM CDDP, followed by 5 μM AZD0156 for another 4 h before a comet assay. Left: the representative comet images are shown. Right, the quantitative data on the length of the tail movement. The values (means ± SD) are from two independent experiments; * p < 0.05 by Student’s t -test ( n = 50). ( B ) Micronucleus frequency in TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ) or LacZ (shLacZ). The cells were treated with 0.5 μM GEM and 5 μM CDDP for 24 h, and were then cultured in the absence or presence of 2.5 μM AZD0156 for another 24 h. Left: representative images of the micronucleus (arrows) are shown. Right, the quantitative data of the percentage of micronuclei. The values (means ± SD) are from two independent experiments; * p < 0.05 by Student’s t -test ( n = 100). ( C ) The colony formation assay was performed in TGBC-24TKB cells receiving shRNAs specific to DNA polymerase θ (shPOLQ) or LacZ (shLacZ). The cells (400 cells/well) were seeded in a six-well plate for 14 days, cultured in the absence or presence of 0.5 μM GEM and 5 μM CCDP for 24 h, and then cultured in the absence or presence of 1 μM AZD0156 for another seven days. Left: representative images are shown. Right: the values (means ± SD) are from two independent experiments; * p < 0.05 by Student’s t -test.

Article Snippet: AZD0156 (ATM inhibitor), gemcitabine, and cisplatin were purchased from AdooQ BioScience (Irvine, CA, USA).

Techniques: Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis, Cell Culture, Colony Assay

Journal: Biomolecules

Article Title: ATM Inhibitor Suppresses Gemcitabine-Resistant BTC Growth in a Polymerase θ Deficiency-Dependent Manner

doi: 10.3390/biom10111529

Figure Lengend Snippet: A diagram illustrating that the ATM inhibitor AZD0156 impairs tumor growth in gemcitabine-resistant or polymerase θ-deficient BTCs.

Article Snippet: AZD0156 (ATM inhibitor), gemcitabine, and cisplatin were purchased from AdooQ BioScience (Irvine, CA, USA).

Techniques: