Journal: Oncotarget

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

doi:

Figure Lengend Snippet: A–F. Pancreatic cancer cell lines were treated with CGK733 for 48 h in a dose dependent manner. Viability was detected by MTS assay. G. PK45-p cells were stained by EthD-III (red) and DAPI (blue) after cells were treated with 20 μM of CGK733 for 24 h. The quantification is shown in the right panel. Bars, SD; *, p < 0.05; ***, p < 0.001.

Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

Techniques: MTS Assay, Staining

Journal: Oncotarget

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

doi:

Figure Lengend Snippet: A. The vesicles were observed under a microscope over a time course, after PK59 cells were treated with 20 μM of CGK733. B. H&E staining was performed after PK59 cells were treated with 20 μM of CGK733 for 24 h. C. PA-Schiff staining was performed after PK59 cells were treated with 20 μM of CGK733 for 24 h. D. Oil red O staining was performed after PK59 cells were treated with 20 μM of CGK733 for 24 h. E. Cells were cultured in the absence of CGK733 for 4 h after cells were treated with 20 μM of CGK733 for 12 h. Arrows indicate the observed vesicles.

Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

Techniques: Microscopy, Staining, Cell Culture

Journal: Oncotarget

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

doi:

Figure Lengend Snippet: A. The expression of ER stress markers were detected by Western blotting after cells were treated with CGK733 for 6 h in a dose dependent manner. B. The vesicles were observed under a microscope after PK45-p and PK59 cells were treated with 20 μM of CGK733 for 12 h and 9 h, respectively. C. and D. Vesiculated cells were quantified in CGK733-treated PK45-p and PK59 cells, respectively. Bars, SD; *** p < 0.001; arrows indicate the observed vesicles.

Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

Techniques: Expressing, Western Blot, Microscopy

Journal: Oncotarget

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

doi:

Figure Lengend Snippet: A. The expression of PERK and CHOP were detected by Western blotting after cells were treated with CGK733 for 6 h in the presence or absence of 1 μM of ionomycin. B. MTS viability assays were performed after cells were treated with CGK733 for 24 h in the presence or absence of 1 μM of ionomycin. C. The vesicles were observed under a microscope after PK45-p and PK59 cells were treated with 10 μM of CGK733 in the presence or absence of 1 μM of ionomycin for 12 h and 9 h, respectively. D. Cal-520 fluorescence combined with bright field microscopy was performed after cells were exposed to 20 μM of CGK733 for 12 h in the presence or absence of 1 μM of ionomycin. Bars, SD; Black arrows indicate the observed vesicles; red arrows indicate the co-localization of calcium ions with the vesicles.

Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

Techniques: Expressing, Western Blot, Microscopy, Fluorescence

Journal: Oncotarget

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

doi:

Figure Lengend Snippet: A. Cells were treated with 20 μM of CGK733 for 6 h after knockdown of CHOP by siRNA for 48 h. B. and C. MTS viability assays were performed after cells were treated with CGK733 for 24 h after knockdown of CHOP by siRNA. D. The vesicles were observed under a microscope after PK45-p and PK59 cells were treated with 20 μM of CGK733 for 12 h and 9 h respectively after knockdown of CHOP by siRNA. Arrows indicate the observed vesicles.

Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

Techniques: Knockdown, Microscopy

Journal: Oncotarget

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

doi:

Figure Lengend Snippet: A. Three downregulated protein spots were identified in the 2-DE gel image of the CGK733-treated cell group compared to the control group. B. MS analysis was performed by the Agilent Spectrum Mill MS proteomics workbench against the Swiss-Prot protein database search engine. The protein numbers are same as in (A).

Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

Techniques: Control

Journal: Oncotarget

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

doi:

Figure Lengend Snippet: A. The expressions of calumenin, protein S100-A11 and CHOP were detected by Western blotting after cells were treated with CGK733 for 6 h in a dose dependent manner. B. The isoforms of protein S100-A11 were detected by 2-D Western blotting after cells were exposed to 20 μM of CGK733. Arrow heads indicate the isoform shown in the 2-DE gel image in Figure . C. Scheme summarizing the CGK733-induced signaling pathway in pancreatic cancer cells.

Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

Techniques: Western Blot

Journal: bioRxiv

Article Title: ATR inhibitors synergize with mitomycin C to enhance cytotoxicity in patient-derived non-muscle invasive bladder cancer organoids

doi: 10.1101/2025.08.29.673024

Figure Lengend Snippet: ( A , B ) BTOR58 organoids were treated for 2h with 0,5 μM gemcitabine (GEM) ( A ) or 0,5 μM epirubicin (EPI) ( B ) alone or in combination with 0,4 μM berzosertib follow-up treatment for 72h. Weekly images were taken for 6 weeks after treatment and representative images at t=1, 3, and 6 weeks are shown. Cell viability assays at t=6 weeks are shown in ( C , D ). ( E , F ) UBTOR8 organoids were treated for 2h with 4 MMC, alone or in combination with 3,2 μM ceralasertib ( E ) or 25 nM tuvusertib ( F ) follow-up treatment for 72h. Representative images at t=1, 3, and 6 weeks after treatment are presented. Cell viability assays at t=6 weeks are shown in ( G , H ). Bar graphs represent mean with SD (n=3).

Article Snippet: Next, PDOs were washed three times with PBS, before BM2+ culture medium was added, and 20h later berzosertib, ceralasertib (TargetMol #T3338), or tuvusertib (TargetMol #T10406) was added for 3 days.

Techniques:

Journal: The Journal of Cell Biology

Article Title: DNA damage promotes microtubule dynamics through a DNA-PK-AKT axis for enhanced repair

doi: 10.1083/jcb.201911025

Figure Lengend Snippet: c-NHEJ pathway is indispensable for DMSR. (A) DMSR was examined when cells were treated with DNA-PK, ATM, or ATR inhibitors. Quantitation of microtubule length is shown ( n > 50). (B) DMSR was examined in cells with depletion of indicated genes. Quantitation of microtubule length is shown ( n > 50). Cells were transfected with an equal amount of scrambled siRNA as an NC. (C) 53BP1 regulated DMSR. DMSR was examined in cells with depletion of indicated genes. Quantitation of microtubule length is shown ( n > 50). (D) Depletion of Ligase 4, XRCC4, or XLF increases the extent of DMSR. Quantitation of microtubule length is shown ( n > 50). (E) Depletion of Ligase 4 prolongs the time course of DMSR. Quantitation of microtubule length is shown ( n > 50). (F) Box plot shows quantitative analysis of GFP-EB3 comet velocity in live siRNA-treated GFP-EB3–expressing G1 RPE-1 cells ( n > 30). (G) Centrosome-dependent microtubule nucleation capacity was determined by relative centrosomal GFP-EB3 intensity in live siRNA-treated GFP-EB3–expressing G1 RPE-1 cells, and quantitative analysis is shown in box plot ( n > 30). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant. A.U., arbitrary units; MT, microtubule; UT, untreated.

Article Snippet: The following chemicals were used: DNA-PKcs inhibitor (MCE; ku57788; 1 μM), ATM inhibitor (MCE; ku55933; 10 μM), ATR inhibitor (MCE; ADZ6783; 1 μM), AKT inhibitor (TargetMol; MK-2206; 5 μM), CHK1 inhibitor (MCE; SB 218078; 10 μM), CHK2 inhibitor (MCE; CCT241533 hydrochloride; 5 μM), PLK1 inhibitor (MCE; BI2536; 100 nM), p38/MAPK inhibitor (MCE; SB 203580; 10 μM), PDK1 inhibitor (MCE; BX-795, 10 μm; BX-912, 10 μM), bleomycin (MCE; 5 μg/ml), mitomycin C (MCE; MMC, 5 μM), camptothecin (Sigma; CPT, 1 μM), cisplatin (Sigma; 2.5 μM), and etoposide (Sigma; 1 μM).

Techniques: Quantitation Assay, Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: DNA damage promotes microtubule dynamics through a DNA-PK-AKT axis for enhanced repair

doi: 10.1083/jcb.201911025

Figure Lengend Snippet: c-NHEJ is required for DMSR. (A) The efficiency of ATR inhibitor or ATM inhibitor was confirmed by probing with pS345-CHK1 antibody or pS1981-ATM antibody, respectively. (B) The time course of DNA-PK activation after RPE-1 cells were exposed to 5 Gy IR in G1/S. DNA-PK activation was determined by immunoblotting with anti-pS2056 DNA-PKcs antibody. (C) Left: Quantitative RT-PCR shows the knockdown efficiency of indicated genes. Right: Western blot analysis shows the efficiency of indicated siRNAs. RPE-1 cells were transfected with indicated siRNAs. Cells were then collected and extracted after 48 h of transfections. The efficiency was measured by indicated antibodies. (D) Depletion of CtIP or other proteins responsible for end resection, such as EXD2, EXO1, and NBS1, at G1 phase does not affect DMSR. U2OS cells were transfected with indicated siRNAs and synchronized at G1/S phase. Cells were then exposed to 2 Gy IR and recovered for 4 h. Left: Western blot analysis shows the efficiency of indicated siRNAs. Right: Microtubule nucleation ability was determined by microtubule regrowth assay. Quantitation of microtubule length was assayed as in . This experiment and the experiment in belong to a same group of experiments. Thus, the same data for the control group was included in this figure and . (E) DMSR is significantly inhibited by depletion of 53BP1 or Ku70. U2OS cells were transfected with indicated siRNA and synchronized at G1/S phase. Cells then were exposed to 2 Gy IR and recovered for 4 h. Microtubules were stained with anti–β-tubulin antibody. Scale bar, 20 µm. (F) Western blot analysis shows the efficiency of indicated siRNAs. (G) Depletion of Ligase 4, XRCC4, or XLF, which are required for c-NHEJ, promote DMSR in U2OS cells. Microtubules (green) were stained with anti–β-tubulin antibody. Scale bar, 20 µm. (H) Western blot analysis shows the efficiency of indicated siRNAs. (I) The activation of DDR and kinetics of DNA damage repair in IR-treated U2OS control cells or siLig4 cells was determined by γH2AX foci formation ( n > 100). ****, P < 0.0001; MT, microtubule; UT, untreated.

Article Snippet: The following chemicals were used: DNA-PKcs inhibitor (MCE; ku57788; 1 μM), ATM inhibitor (MCE; ku55933; 10 μM), ATR inhibitor (MCE; ADZ6783; 1 μM), AKT inhibitor (TargetMol; MK-2206; 5 μM), CHK1 inhibitor (MCE; SB 218078; 10 μM), CHK2 inhibitor (MCE; CCT241533 hydrochloride; 5 μM), PLK1 inhibitor (MCE; BI2536; 100 nM), p38/MAPK inhibitor (MCE; SB 203580; 10 μM), PDK1 inhibitor (MCE; BX-795, 10 μm; BX-912, 10 μM), bleomycin (MCE; 5 μg/ml), mitomycin C (MCE; MMC, 5 μM), camptothecin (Sigma; CPT, 1 μM), cisplatin (Sigma; 2.5 μM), and etoposide (Sigma; 1 μM).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Knockdown, Transfection, Regrowth Assay, Quantitation Assay, Control, Staining

Journal: eLife

Article Title: Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations

doi: 10.7554/eLife.32222

Figure Lengend Snippet: ( A ) Amino acid sequence 408 through 420 of ARP8. The Ser412 residue, within the ATM/ATR substrate motif and the CK2 substrate motif, is indicated. ( B ) Immunoprecipitation analysis of ARP8 phosphorylation. U2OS cells transiently expressing an empty HA vector or a vector encoding HA-tagged ARP8 were treated with DMSO (ctrl) or etoposide (Etp) for 15 min, then washed twice and cultured in complete medium for the indicated times. The nuclear extracts were incubated with anti-HA-conjugated anti-mouse IgG Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with an anti-ATM/ATR substrate antibody or an anti-HA antibody. λPPase treatment identified the band of phosphorylated HA-ARP8. The blot of input was probed by antibodies against phospho-ATM (p-ATM), γ H2AX or phospho-RPA2 at Ser4/8 (p-RPA2). β-actin was used as a loading control. ( C ) Identification of the ARP8 phosphorylation site by an immunoprecipitation analysis. U2OS cells were transfected with an empty HA vector (vet), or a vector encoding HA-tagged wild-type ARP8 (WT) or HA-ARP8 S412A (S412A) for 48 hr. The cells were washed after treatment with etoposide or DMSO for 15 min, cultured in fresh medium, and harvested at the indicated time points. Whole cell extracts were used for the immunoprecipitation analysis. ( D ) Etoposide-induced ARP8 phosphorylation in ATM-deficient BIVA and ATM-proficient 11–4 cells. Immunoprecipitation analysis of cell extracts of BIVA or 11–4 cells transfected with HA-tagged wild-type ARP8 using anti-HA antibodies. The cells were treated with DMSO (ctrl) or etoposide (Etp) for 15 min, cultured in fresh medium, and harvested at the indicated time points. Whole cell extracts were used for the immunoprecipitation analysis, which was performed as described in ( B ). The amounts of phosphorylated ARP8 and HA-ARP8 were quantified, using the Image J software. The results of the quantitative analysis are shown as the relative values to the DMSO controls. Source data are presented in . ( E ) Immunoprecipitation analysis of cell extracts from 11 to 4 cells expressing HA-tagged ARP8. The cells were treated with DMSO, 10 μM ATMi (KU55933), or 10 μM ATRi (VE821) for 2 hr before etoposide treatment, and then the inhibitors (5 μM) were added after the cells were washed. 10.7554/eLife.32222.005 Figure 1—source data 1. Source raw data for .

Article Snippet: Unless otherwise stated, both the ATM inhibitor KU55933 (Merck Millipore, Billerica, USA) and ATR inhibitor IV VE821 (Merck Millipore) were used at 10 μM for two hours before etoposide treatment and at 5 μM after the cells were washed, respectively.

Techniques: Sequencing, Residue, Immunoprecipitation, Phospho-proteomics, Expressing, Plasmid Preparation, Cell Culture, Incubation, Western Blot, Control, Transfection, Software

Journal: eLife

Article Title: Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations

doi: 10.7554/eLife.32222

Figure Lengend Snippet: ( A ) ChIP analysis of the RAD51 loading onto the MLL BCR in ATMi or ATRi or a combination of ATMi and ATRi treated 11–4 cells. 11–4 cells were treated with ATMi (10 µM), ATRi (10 µM), or a combination of ATMi and ATRi for 2 hr before etoposide treatment. After washing the cells, the inhibitors (5 μM) were added until the cells were harvested. The ChIP analysis was performed as described in . Values represent the means ± SE from three independent experiments. *p<0.05. **p<0.01. Source data are presented in . ( B ) The percentages of 11–4 cells with split chromosome 11q23 signals are shown. 11–4 cells were treated with ATMi (10 µM), ATRi (10 µM), or a combination of ATMi and ATRi for 2 hr before etoposide treatment. After the cells were washed, the inhibitors (5 μM) were added until the cells were harvested. Dual-color FISH analyses of chromosome 11q23 were performed as described in . Values represent the means ± SE from three independent experiments. *p<0.05. **p<0.01, ***p<0.001, n.s.: no significant difference. 10.7554/eLife.32222.026 Figure 6—source data 1. Source raw data for .

Article Snippet: Unless otherwise stated, both the ATM inhibitor KU55933 (Merck Millipore, Billerica, USA) and ATR inhibitor IV VE821 (Merck Millipore) were used at 10 μM for two hours before etoposide treatment and at 5 μM after the cells were washed, respectively.

Techniques: