Journal: bioRxiv

Article Title: Genomic and transcriptomic profiling of high-risk bladder cancer reveals diverse molecular and microenvironment ecosystems

doi: 10.1101/2024.12.21.629010

Figure Lengend Snippet: A. FGFR3 mRNA levels across DepMap cell lines classified as having high or low expression of Subtype 4 gene program markers. (p-value was calculated from a two-sided Kruskal-Wallis test) B. Volcano plot showing differences in IC50 between cell-lines with high vs low Subtype 4 gene program score on the x-axis and -Log10 adjusted p-value for each comparison on the y-axis. Each point represents a unique chemical perturbation (Left). Drugs with increased sensitivity in cell-lines that have high expression of Subtype 4 gene program (Right). C. MYC and FGFR3 protein levels in UMUC3 or UMUC9 cell-lines overexpressing GFP controls or FGFR3 S249C mutations. (Vinculin was used as loading control for the cytoplasmic fraction, RPA32 was used as loading control for the nuclear fraction) (n=2) D. Survival curves for UMUC3 and UMUC9 cell-lines overexpressing GFP controls or FGFR3 S249C mutations treated with MEK inhibitor Trametinib or MYC inhibitor for 7 days. (n=3).

Article Snippet: Protein expression was verified using western blotting with these antibodies: anti-ERBB2 (Cell Signaling Technologies, 2165), anti-FGFR3 (Cell Signaling Technologies, 4574), anti-RPA32 (Cell Signaling Technologies, 35869), anti-Tubulin (Sigma, T5168), anti-Vinculin (Invitrogen, MA5-11690), anti- MYC (Invitrogen, 13-2500).

Techniques: Expressing, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: EtOAc does not affect DNA damage repair ability induced by camptothecin. ( A ) RPA32 S4/8 foci intensity in HeLa cells treated for one hour with EtOAc at 0.63 μg/mL, followed by incubation with 1 μM CPT for an additional two hours. Foci number was measured with Fiji software. **** p < 0.0001. ( B ) Representative images of pRPA32 following incubation with the indicated reagents. All immunofluorescence images were acquired with the LSM900 using AiryScan2. Magnification 63×. ( C ) γH2AX intensity was measured in HeLa cells pre-treated with EtOAc extract or DMSO for one hour, followed by 1 μM CPT treatment. At the end of incubation time we performed a drugs washout, monitoring the DNA repair as a measure of γH2AX nuclear signal. Immunofluorescence images were analyzed with Fiji software. * p < 0.05 and **** p < 0.0001. ( D ) Representative images of H2AX S139 nuclear intensity in HeLa cells treated with 1 μM CPT and DMSO for three hours, followed by drug washout to monitor H2AX recovery. Magnification 63×. ( E ) Immunofluorescence analysis of HeLa cells treated for one hour with EtOAc at 0.63 μg/mL, followed by incubation with 1 μM CPT for an additional two hours. Magnification 63×. ( F ) HeLa cells stably transfected with the pDR-GFP vector were co-transfected with I-SceI endonuclease and incubated with 0.63 μg/mL of EtOAc extract for 24 h. HR activity was measured using FACS analysis, with GFP levels serving as an indicator of HR frequency. Data are presented as means ± standard deviation from three independent experiments. Statistically significant differences are indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Cells were then incubated for 1 h at 37 °C with primary antibodies: anti-RAD51 (1:200, sc-8349, Santa Cruz Biotechnology) anti-RPA32 S4/8 (1:200, A300-245A, Bethyl Laboratories), anti-ERCC1 (1:200, NB500-704, Novus Biological, Centennial, CO, USA), γH2AX S-139 (1:200, ab2893, Abcam, Cambridge, UK), and γH2AX S-139 (1:200, 05-636, Millipore, Burlington, MA, USA).

Techniques: Incubation, Software, Immunofluorescence, Stable Transfection, Transfection, Plasmid Preparation, Activity Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: OA treatment does not affect the DNA repair ability compared to CPT alone. ( A ) The neutral comet assay was performed as described in . HeLa cells were pre-treated with OA or DMSO, followed by incubation with 1 μM CPT for an additional two hours. At the end of the incubation period, a drug washout was performed to monitor DNA repair. For each condition, 5 × 10⁴ cells were spotted onto glass slides and stained with SYBR Green. We analyzed 30 cells for each condition. The results show the means and standard deviation (SD) of three independent experiments. Statistically significant differences are indicated by * p < 0.05, ** p < 0.01, and **** p < 0.0001. ( B ) Representative images of the neutral comet assay. Magnification 63×. ( C ) Immunofluorescence analysis of pRPA32 S4/8 foci per cell in HeLa cells treated with CPT alone or in combination with OA. Immunofluorescence staining was performed with pRPA32 S4/8, and Hoechst was used as a DNA marker. We analyzed 30 cells for each condition across three independent experiments. Statistical analysis was performed using a t -test, and significant differences are indicated by **** p < 0.0001. ( D ) Representative images of pRPA32 S4/8 in HeLa cells treated as described in ( C ). Magnification 63×. ( E ) HeLa cells were pre-treated with 0.63 µg/mL of OA for one hour, followed by incubation with 1 µM CPT for an additional two hours. Chromatin-enriched purification was performed to separate soluble (S) and chromatin-bound (P) fractions. RPA32 protein was used as a marker for the soluble fraction and as a control for DNA damage, while Lamin A/C served as a loading control for the chromatin fraction.

Article Snippet: Cells were then incubated for 1 h at 37 °C with primary antibodies: anti-RAD51 (1:200, sc-8349, Santa Cruz Biotechnology) anti-RPA32 S4/8 (1:200, A300-245A, Bethyl Laboratories), anti-ERCC1 (1:200, NB500-704, Novus Biological, Centennial, CO, USA), γH2AX S-139 (1:200, ab2893, Abcam, Cambridge, UK), and γH2AX S-139 (1:200, 05-636, Millipore, Burlington, MA, USA).

Techniques: Neutral Comet Assay, Incubation, Staining, SYBR Green Assay, Standard Deviation, Immunofluorescence, Marker, Purification, Control

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: ERCC1 localized to DNA damaged sites upon OA treatment. ( A ) HeLa cells were pre-treated with Oleanolic Acid (OA) at 0.63 µg/mL or DMSO for one hour, followed by incubation with 1 µM CPT for two hours. Chromatin purification was performed to separate soluble (S) and chromatin-bound (P) fractions. Total RPA32 was used for normalization of the soluble fraction (S) and as a DNA damage control, while Lamin A/C served as a loading control for the chromatin fraction. ( B ) Immunofluorescence analysis of HeLa cells treated as described in ( A ) was conducted to assess ERCC1 foci formation. Foci were quantified using Fiji software. The graph shows means ± standard deviation (SD) from three independent experiments, with 30 cells analyzed per condition. Statistical significance was determined by Student’s t -test, with results indicated as **** p < 0.0001. ( C ) Representative images of ERCC1 foci from HeLa cells treated as described in ( A ) are shown. Images were acquired using Zen software. Hoechst staining was used as a DNA marker to visualize cell nuclei. Magnification 63×. ( D ) Immunofluorescence images showing the colocalization of ERCC1 and γH2AX in HeLa cells treated as in ( A ). Colocalization masks were generated using Zen software to illustrate the overlap between ERCC1 and γH2AX foci. ( E ) Pearson’s correlation coefficient was calculated using Zen software to quantify the colocalization of ERCC1 and γH2AX in HeLa cells treated as described in ( A ). The graph shows means ± SD from three independent experiments, with 30 cells analyzed per condition. **** p < 0.0001. ( F ) A scatter plot depicting ERCC1 and γH2AX colocalization in HeLa cells treated with OA or DMSO for one hour, followed by CPT treatment, is presented. Images were analyzed with Zen software to visualize the distribution and overlap of ERCC1 and γH2AX foci.

Article Snippet: Cells were then incubated for 1 h at 37 °C with primary antibodies: anti-RAD51 (1:200, sc-8349, Santa Cruz Biotechnology) anti-RPA32 S4/8 (1:200, A300-245A, Bethyl Laboratories), anti-ERCC1 (1:200, NB500-704, Novus Biological, Centennial, CO, USA), γH2AX S-139 (1:200, ab2893, Abcam, Cambridge, UK), and γH2AX S-139 (1:200, 05-636, Millipore, Burlington, MA, USA).

Techniques: Incubation, Purification, Control, Immunofluorescence, Software, Standard Deviation, Staining, Marker, Generated

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: EtOAc does not affect DNA damage repair ability induced by camptothecin. ( A ) RPA32 S4/8 foci intensity in HeLa cells treated for one hour with EtOAc at 0.63 μg/mL, followed by incubation with 1 μM CPT for an additional two hours. Foci number was measured with Fiji software. **** p < 0.0001. ( B ) Representative images of pRPA32 following incubation with the indicated reagents. All immunofluorescence images were acquired with the LSM900 using AiryScan2. Magnification 63×. ( C ) γH2AX intensity was measured in HeLa cells pre-treated with EtOAc extract or DMSO for one hour, followed by 1 μM CPT treatment. At the end of incubation time we performed a drugs washout, monitoring the DNA repair as a measure of γH2AX nuclear signal. Immunofluorescence images were analyzed with Fiji software. * p < 0.05 and **** p < 0.0001. ( D ) Representative images of H2AX S139 nuclear intensity in HeLa cells treated with 1 μM CPT and DMSO for three hours, followed by drug washout to monitor H2AX recovery. Magnification 63×. ( E ) Immunofluorescence analysis of HeLa cells treated for one hour with EtOAc at 0.63 μg/mL, followed by incubation with 1 μM CPT for an additional two hours. Magnification 63×. ( F ) HeLa cells stably transfected with the pDR-GFP vector were co-transfected with I-SceI endonuclease and incubated with 0.63 μg/mL of EtOAc extract for 24 h. HR activity was measured using FACS analysis, with GFP levels serving as an indicator of HR frequency. Data are presented as means ± standard deviation from three independent experiments. Statistically significant differences are indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following antibodies were used: RPA32 (1:5000, A300–244A, Bethyl Laboratories, Montgomery, TX, USA), RPA32 S4/S8 (1:2000, A300–245A, Bethyl Laboratories), Lamin A/C (1:1000, #4777, Cell Signalling, Danvers, MA, USA), CHK1 S345 (1:1000, #2348, Cell Signalling), CHK1 (1:1000, #2360, Cell Signalling), GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA), RAD51 (1:1000, NB100-148, Novus Biologicals, Centennial, CO, USA), and KU70 (1:500, sc-1486, Santa Cruz Biotechnology).

Techniques: Incubation, Software, Immunofluorescence, Stable Transfection, Transfection, Plasmid Preparation, Activity Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: OA treatment does not affect the DNA repair ability compared to CPT alone. ( A ) The neutral comet assay was performed as described in . HeLa cells were pre-treated with OA or DMSO, followed by incubation with 1 μM CPT for an additional two hours. At the end of the incubation period, a drug washout was performed to monitor DNA repair. For each condition, 5 × 10⁴ cells were spotted onto glass slides and stained with SYBR Green. We analyzed 30 cells for each condition. The results show the means and standard deviation (SD) of three independent experiments. Statistically significant differences are indicated by * p < 0.05, ** p < 0.01, and **** p < 0.0001. ( B ) Representative images of the neutral comet assay. Magnification 63×. ( C ) Immunofluorescence analysis of pRPA32 S4/8 foci per cell in HeLa cells treated with CPT alone or in combination with OA. Immunofluorescence staining was performed with pRPA32 S4/8, and Hoechst was used as a DNA marker. We analyzed 30 cells for each condition across three independent experiments. Statistical analysis was performed using a t -test, and significant differences are indicated by **** p < 0.0001. ( D ) Representative images of pRPA32 S4/8 in HeLa cells treated as described in ( C ). Magnification 63×. ( E ) HeLa cells were pre-treated with 0.63 µg/mL of OA for one hour, followed by incubation with 1 µM CPT for an additional two hours. Chromatin-enriched purification was performed to separate soluble (S) and chromatin-bound (P) fractions. RPA32 protein was used as a marker for the soluble fraction and as a control for DNA damage, while Lamin A/C served as a loading control for the chromatin fraction.

Article Snippet: The following antibodies were used: RPA32 (1:5000, A300–244A, Bethyl Laboratories, Montgomery, TX, USA), RPA32 S4/S8 (1:2000, A300–245A, Bethyl Laboratories), Lamin A/C (1:1000, #4777, Cell Signalling, Danvers, MA, USA), CHK1 S345 (1:1000, #2348, Cell Signalling), CHK1 (1:1000, #2360, Cell Signalling), GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA), RAD51 (1:1000, NB100-148, Novus Biologicals, Centennial, CO, USA), and KU70 (1:500, sc-1486, Santa Cruz Biotechnology).

Techniques: Neutral Comet Assay, Incubation, Staining, SYBR Green Assay, Standard Deviation, Immunofluorescence, Marker, Purification, Control

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: ERCC1 localized to DNA damaged sites upon OA treatment. ( A ) HeLa cells were pre-treated with Oleanolic Acid (OA) at 0.63 µg/mL or DMSO for one hour, followed by incubation with 1 µM CPT for two hours. Chromatin purification was performed to separate soluble (S) and chromatin-bound (P) fractions. Total RPA32 was used for normalization of the soluble fraction (S) and as a DNA damage control, while Lamin A/C served as a loading control for the chromatin fraction. ( B ) Immunofluorescence analysis of HeLa cells treated as described in ( A ) was conducted to assess ERCC1 foci formation. Foci were quantified using Fiji software. The graph shows means ± standard deviation (SD) from three independent experiments, with 30 cells analyzed per condition. Statistical significance was determined by Student’s t -test, with results indicated as **** p < 0.0001. ( C ) Representative images of ERCC1 foci from HeLa cells treated as described in ( A ) are shown. Images were acquired using Zen software. Hoechst staining was used as a DNA marker to visualize cell nuclei. Magnification 63×. ( D ) Immunofluorescence images showing the colocalization of ERCC1 and γH2AX in HeLa cells treated as in ( A ). Colocalization masks were generated using Zen software to illustrate the overlap between ERCC1 and γH2AX foci. ( E ) Pearson’s correlation coefficient was calculated using Zen software to quantify the colocalization of ERCC1 and γH2AX in HeLa cells treated as described in ( A ). The graph shows means ± SD from three independent experiments, with 30 cells analyzed per condition. **** p < 0.0001. ( F ) A scatter plot depicting ERCC1 and γH2AX colocalization in HeLa cells treated with OA or DMSO for one hour, followed by CPT treatment, is presented. Images were analyzed with Zen software to visualize the distribution and overlap of ERCC1 and γH2AX foci.

Article Snippet: The following antibodies were used: RPA32 (1:5000, A300–244A, Bethyl Laboratories, Montgomery, TX, USA), RPA32 S4/S8 (1:2000, A300–245A, Bethyl Laboratories), Lamin A/C (1:1000, #4777, Cell Signalling, Danvers, MA, USA), CHK1 S345 (1:1000, #2348, Cell Signalling), CHK1 (1:1000, #2360, Cell Signalling), GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA), RAD51 (1:1000, NB100-148, Novus Biologicals, Centennial, CO, USA), and KU70 (1:500, sc-1486, Santa Cruz Biotechnology).

Techniques: Incubation, Purification, Control, Immunofluorescence, Software, Standard Deviation, Staining, Marker, Generated

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: EtOAc does not affect DNA damage repair ability induced by camptothecin. ( A ) RPA32 S4/8 foci intensity in HeLa cells treated for one hour with EtOAc at 0.63 μg/mL, followed by incubation with 1 μM CPT for an additional two hours. Foci number was measured with Fiji software. **** p < 0.0001. ( B ) Representative images of pRPA32 following incubation with the indicated reagents. All immunofluorescence images were acquired with the LSM900 using AiryScan2. Magnification 63×. ( C ) γH2AX intensity was measured in HeLa cells pre-treated with EtOAc extract or DMSO for one hour, followed by 1 μM CPT treatment. At the end of incubation time we performed a drugs washout, monitoring the DNA repair as a measure of γH2AX nuclear signal. Immunofluorescence images were analyzed with Fiji software. * p < 0.05 and **** p < 0.0001. ( D ) Representative images of H2AX S139 nuclear intensity in HeLa cells treated with 1 μM CPT and DMSO for three hours, followed by drug washout to monitor H2AX recovery. Magnification 63×. ( E ) Immunofluorescence analysis of HeLa cells treated for one hour with EtOAc at 0.63 μg/mL, followed by incubation with 1 μM CPT for an additional two hours. Magnification 63×. ( F ) HeLa cells stably transfected with the pDR-GFP vector were co-transfected with I-SceI endonuclease and incubated with 0.63 μg/mL of EtOAc extract for 24 h. HR activity was measured using FACS analysis, with GFP levels serving as an indicator of HR frequency. Data are presented as means ± standard deviation from three independent experiments. Statistically significant differences are indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following antibodies were used: RPA32 (1:5000, A300–244A, Bethyl Laboratories, Montgomery, TX, USA), RPA32 S4/S8 (1:2000, A300–245A, Bethyl Laboratories), Lamin A/C (1:1000, #4777, Cell Signalling, Danvers, MA, USA), CHK1 S345 (1:1000, #2348, Cell Signalling), CHK1 (1:1000, #2360, Cell Signalling), GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA), RAD51 (1:1000, NB100-148, Novus Biologicals, Centennial, CO, USA), and KU70 (1:500, sc-1486, Santa Cruz Biotechnology).

Techniques: Incubation, Software, Immunofluorescence, Stable Transfection, Transfection, Plasmid Preparation, Activity Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: OA treatment does not affect the DNA repair ability compared to CPT alone. ( A ) The neutral comet assay was performed as described in . HeLa cells were pre-treated with OA or DMSO, followed by incubation with 1 μM CPT for an additional two hours. At the end of the incubation period, a drug washout was performed to monitor DNA repair. For each condition, 5 × 10⁴ cells were spotted onto glass slides and stained with SYBR Green. We analyzed 30 cells for each condition. The results show the means and standard deviation (SD) of three independent experiments. Statistically significant differences are indicated by * p < 0.05, ** p < 0.01, and **** p < 0.0001. ( B ) Representative images of the neutral comet assay. Magnification 63×. ( C ) Immunofluorescence analysis of pRPA32 S4/8 foci per cell in HeLa cells treated with CPT alone or in combination with OA. Immunofluorescence staining was performed with pRPA32 S4/8, and Hoechst was used as a DNA marker. We analyzed 30 cells for each condition across three independent experiments. Statistical analysis was performed using a t -test, and significant differences are indicated by **** p < 0.0001. ( D ) Representative images of pRPA32 S4/8 in HeLa cells treated as described in ( C ). Magnification 63×. ( E ) HeLa cells were pre-treated with 0.63 µg/mL of OA for one hour, followed by incubation with 1 µM CPT for an additional two hours. Chromatin-enriched purification was performed to separate soluble (S) and chromatin-bound (P) fractions. RPA32 protein was used as a marker for the soluble fraction and as a control for DNA damage, while Lamin A/C served as a loading control for the chromatin fraction.

Article Snippet: The following antibodies were used: RPA32 (1:5000, A300–244A, Bethyl Laboratories, Montgomery, TX, USA), RPA32 S4/S8 (1:2000, A300–245A, Bethyl Laboratories), Lamin A/C (1:1000, #4777, Cell Signalling, Danvers, MA, USA), CHK1 S345 (1:1000, #2348, Cell Signalling), CHK1 (1:1000, #2360, Cell Signalling), GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA), RAD51 (1:1000, NB100-148, Novus Biologicals, Centennial, CO, USA), and KU70 (1:500, sc-1486, Santa Cruz Biotechnology).

Techniques: Neutral Comet Assay, Incubation, Staining, SYBR Green Assay, Standard Deviation, Immunofluorescence, Marker, Purification, Control

Journal: International Journal of Molecular Sciences

Article Title: Oleanolic Acid Modulates DNA Damage Response to Camptothecin Increasing Cancer Cell Death

doi: 10.3390/ijms252413475

Figure Lengend Snippet: ERCC1 localized to DNA damaged sites upon OA treatment. ( A ) HeLa cells were pre-treated with Oleanolic Acid (OA) at 0.63 µg/mL or DMSO for one hour, followed by incubation with 1 µM CPT for two hours. Chromatin purification was performed to separate soluble (S) and chromatin-bound (P) fractions. Total RPA32 was used for normalization of the soluble fraction (S) and as a DNA damage control, while Lamin A/C served as a loading control for the chromatin fraction. ( B ) Immunofluorescence analysis of HeLa cells treated as described in ( A ) was conducted to assess ERCC1 foci formation. Foci were quantified using Fiji software. The graph shows means ± standard deviation (SD) from three independent experiments, with 30 cells analyzed per condition. Statistical significance was determined by Student’s t -test, with results indicated as **** p < 0.0001. ( C ) Representative images of ERCC1 foci from HeLa cells treated as described in ( A ) are shown. Images were acquired using Zen software. Hoechst staining was used as a DNA marker to visualize cell nuclei. Magnification 63×. ( D ) Immunofluorescence images showing the colocalization of ERCC1 and γH2AX in HeLa cells treated as in ( A ). Colocalization masks were generated using Zen software to illustrate the overlap between ERCC1 and γH2AX foci. ( E ) Pearson’s correlation coefficient was calculated using Zen software to quantify the colocalization of ERCC1 and γH2AX in HeLa cells treated as described in ( A ). The graph shows means ± SD from three independent experiments, with 30 cells analyzed per condition. **** p < 0.0001. ( F ) A scatter plot depicting ERCC1 and γH2AX colocalization in HeLa cells treated with OA or DMSO for one hour, followed by CPT treatment, is presented. Images were analyzed with Zen software to visualize the distribution and overlap of ERCC1 and γH2AX foci.

Article Snippet: The following antibodies were used: RPA32 (1:5000, A300–244A, Bethyl Laboratories, Montgomery, TX, USA), RPA32 S4/S8 (1:2000, A300–245A, Bethyl Laboratories), Lamin A/C (1:1000, #4777, Cell Signalling, Danvers, MA, USA), CHK1 S345 (1:1000, #2348, Cell Signalling), CHK1 (1:1000, #2360, Cell Signalling), GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA), RAD51 (1:1000, NB100-148, Novus Biologicals, Centennial, CO, USA), and KU70 (1:500, sc-1486, Santa Cruz Biotechnology).

Techniques: Incubation, Purification, Control, Immunofluorescence, Software, Standard Deviation, Staining, Marker, Generated

Journal: Neoplasia (New York, N.Y.)

Article Title: Suppression of ADP-ribosylation reversal triggers cell vulnerability to alkylating agents

doi: 10.1016/j.neo.2024.101092

Figure Lengend Snippet:

Article Snippet: In this study, the following antibodies were used: anti-poly/mono ADPr (rabbit monoclonal), Cell Signaling, Cat. 83732; anti-H2AX (rabbit polyclonal), Cell Signaling, Cat. 2595; anti-PARP1 (rabbit monoclonal), Abcam, Cat. ab32138; anti-PARP1 (mouse monoclonal); BD Biosciences, Cat. 556494; anti-γH2AX (mouse monoclonal), Cell Signaling, Cat. D7T2V; anti-α-tubulin (mouse monoclonal), Sigma-Aldrich, Cat. T6074; anti-β-tubulin (rabbit polyclonal), Abcam, Cat. ab6046; anti-Phospho-RPA32/RPA2 (Ser8) (rabbit polyclonal), Cell Signaling, Cat. 54762; anti-RPA32 p-S4/8 (rabbit polyclonal), Bethyl, Cat. A300-245A; anti-RPA32 (rabbit polyclonal), Bethyl, Cat. A300-244A; RPA32/RPA2 (rabbit polyclonal) Cell Signaling, Cat. 52448; anti-PAN-ADP-RIBOSE binding reagent, Merck Millipore, Cat. MABE1016; anti-Poly(ADP-ribose) (rabbit polyclonal), Enzo Life Sciences, Cat. ALX-210-890A-0100; anti-ADPRHL2 (rabbit monoclonal), Sigma-Aldrich, Cat. HPA027104; anti-histone H3 (rabbit polyclonal), Millipore, Cat. 07–690; anti-p53 (mouse monoclonal), Santa Cruz, Cat. sc-126; anti-p53 K370ac, Cell Signaling; anti-p53 K382ac, Cell Signaling, Cat. 2525; anti-Caspase 3 (rabbit monoclonal), Cell Signaling, Cat. 14220; anti-Caspase 7 (rabbit monoclonal); Cell Signaling, Cat. 12827; anti-HPF1/C4orf27 (rabbit polyclonal), NovusBio; Cat. NBP1-93973; anti-H2A (rabbit polyclonal), Abcam, Cat. ab18255; anti-PARP2 (mouse monoclonal), Millipore, Cat. MABE18; anti-lamin A (rabbit polyclonal), Abcam, Cat. ab26300;

Techniques: Binding Assay, Recombinant, Protease Inhibitor, Transfection, Proliferation Assay, Flow Cytometry, Fractionation, Cell Culture, Plasmid Preparation, Software, Microscopy