Journal: Molecular Cancer Research

Article Title: Inhibition of Expression of the Chromatin Remodeling Gene, SNF2L, Selectively Leads to DNA Damage, Growth Inhibition, and Cancer Cell Death

doi: 10.1158/1541-7786.mcr-09-0119

Figure Lengend Snippet: FIGURE 4. Knockdown of SNF2L caused DNA damage and resulted in a DNA damage response in the HM lines. A. MDA-MB-468 cells were transfected with SNF2L siRNA or NCSI. Forty-eight hours after transfection, DNA damage was analyzed by the Comet assay and the results showed damaged DNA (the comet tail) outside the nucleus after treatment of SNF2L siRNA (bottom) compared with undamaged DNA in the cells treated with NCSI (top). B. The per- centage of cells with damaged DNA is depicted in representative HM, LG, and NU lines. There was a marked increase in DNA damage in the HM lines. C. The MDA-MB-468 cells that had been so treated were also analyzed for the levels of both total and phosphorylated proteins involved in a DNA damage response, including ATR, BRCA1, CHK1, CHK2, and H2AX. Increased phosphorylation of all these proteins was observed with SNF2L knockdown but no alterations of the respective total proteins.

Article Snippet: Antibodies used for detection of DNA damage were obtained from the DNA Damage Antibody Sampler (Cell Signaling Technology, Inc.), which included Phospho-ATR (Ser428), Phospho-ATM (Ser1981) mouse monoclonal antibody (mAb), Phospho-BRCA1 (Ser1524), Phospho-CHK1 (Ser296), Phospho-CHK2 (Thr68), PhosphoHistone H2AX (Ser139), and Phospho-p53 (Ser15; 16G8) mouse mAb. p53 (7F5) rabbit mAb and β-actin (13E5) rabbit mAb were also used (Cell Signaling Technology, Inc.).

Techniques: Knockdown, Transfection, Single Cell Gel Electrophoresis, Phospho-proteomics

Journal: Molecular Cancer Research

Article Title: Inhibition of Expression of the Chromatin Remodeling Gene, SNF2L, Selectively Leads to DNA Damage, Growth Inhibition, and Cancer Cell Death

doi: 10.1158/1541-7786.mcr-09-0119

Figure Lengend Snippet: FIGURE 7. DNA damage, DNA damage response, and apoptosis. A. Flow cytometry of cells stained in Annexin-V and PI solution and treated with NCSI (left), SNF2L siRNA (center), and SNF2L siRNA with a general caspase inhibitor (right). Only with SNF2L knockdown alone was there an increase in both early (Annexin-V) and late (PI) apoptosis (top right). Inhibition of apoptosis abolished this effect (bottom) and the control negative siRNA (NCSI) produced no increased apoptosis (top left). B. Comet assay confirmed that DNA damage was induced by SNF2L knockdown (center) and was not observed when the negative control (NCSI) was used (left). Significantly, DNA damage still occurred even when apoptosis was inhibited with a general caspase inhibitor (right). C. Western blot indicated that DNA damage and the resultant DNA damage response, e.g., p-H2AX from SNF2L knockdown preceded and did not follow apoptosis. SNF2L siRNA, but not NCSI, increased cleaved PARP (a surrogate of apoptosis) and p-H2AX. Specific caspase inhibitors to caspase 3, caspase 8, and caspase 9 were only minimally effective at inhibiting cleaved PARP. The general caspase inhibitor I, on the other hand, showed significant inhibition of PARP cleavage (apoptosis), yet p-H2AX was still expressed.

Article Snippet: Antibodies used for detection of DNA damage were obtained from the DNA Damage Antibody Sampler (Cell Signaling Technology, Inc.), which included Phospho-ATR (Ser428), Phospho-ATM (Ser1981) mouse monoclonal antibody (mAb), Phospho-BRCA1 (Ser1524), Phospho-CHK1 (Ser296), Phospho-CHK2 (Thr68), PhosphoHistone H2AX (Ser139), and Phospho-p53 (Ser15; 16G8) mouse mAb. p53 (7F5) rabbit mAb and β-actin (13E5) rabbit mAb were also used (Cell Signaling Technology, Inc.).

Techniques: Flow Cytometry, Staining, Knockdown, Inhibition, Control, Produced, Single Cell Gel Electrophoresis, Negative Control, Western Blot

Journal: Molecular human reproduction

Article Title: Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome.

doi: 10.1093/molehr/gaw065

Figure Lengend Snippet: Figure 2 Effect of S1P on the expression of endothelial adherens junction proteins in endothelial cells in the presence of FF from controls and patients at risk of OHSS. Densitometric quantification of (A) phospho-vascular endothelial (p-VE)-cadherin, (B) VE-cadherin, (C) N-cadherin and (D) β-catenin. Representative immunoblots are shown in the lower panel. Data are expressed as means ± SEM normalized to β-actin of three independent experiments using 20 control patients and 20 OHSS patients at risk of OHSS (*P < 0.05).

Article Snippet: The blot was preincubated in blocking buffer (5% nonfat milk, 0.05% Tween 20 in 20 mM TBS pH 8.0) for 1 h at room temperature and incubated overnight in blocking buffer at 4°C with appropriate primary antibodies: β-actin 1/3000 (sc-1616-R), N-cadherin 1/200 (sc-7939), vascular endothelial (VE)-cadherin 1/100 (sc-9989) and β-catenin 1/200 (sc-59737) (Santa Cruz Biotechnology, Inc., Santa Cruz, USA), phospho-VE-cadherin 1/1000 (MAB1002) (Millipore, MA, USA), VEGF 1/1000 (ab46154) (Abcam, Cambridge, USA), KDR (VEGFR-2) 1/1000 (D5B1) (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal: iScience

Article Title: IGFBP3 promotes resistance to Olaparib via modulating EGFR signaling in advanced prostate cancer

doi: 10.1016/j.isci.2024.108984

Figure Lengend Snippet: EGFR/DNAPKcs-mediated DSB NHEJ signaling upregulated in Olaparib-resistant prostate cancer (A) The protein expression of phospho-EGF receptor (Tyr1068) and IGFBP3 in parental cells, OlapR cells was determined by western blot analysis. Tubulin served as a loading control. (B) The protein expression of phospho-DNA-PKcs (Ser2056) and IGFBP3 in parental cells, OlapR cells was determined by western blot analysis. Tubulin served as a loading control. (C) The cell growth of 2B-OlapR cells transfected with EGFR siRNA for 3 days and 5 days was determined by CCK-8. (D) In the cBioPortal database (SUC2/PCF Dream Team, PNAS 2019), EGFR gene expression levels were determined in tumor samples of different Gleason scores. (E) In the cBioPortal database (Broad/Cornell, Nat Genetics 2012), PRKDC (DNA-PKcs) gene expression levels were determined in tumor samples of different Gleason scores. (F) In the cBioPortal database (MSK, Science 2022), PRKDC (DNA-PKcs) gene expression levels were determined in organoid derived cancer types, prostate adenocarcinoma and prostate. p value less than 0.05 was considered significant (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001). Statistical analysis was performed using Student’s t test.

Article Snippet: Phospho-DNA-PKcs (Ser2056) , Cell Signaling Technology , Cat#: 68716, RRID: AB_2939025.

Techniques: Expressing, Western Blot, Control, Transfection, CCK-8 Assay, Gene Expression, Derivative Assay

Journal: iScience

Article Title: IGFBP3 promotes resistance to Olaparib via modulating EGFR signaling in advanced prostate cancer

doi: 10.1016/j.isci.2024.108984

Figure Lengend Snippet: IGFBP3 and EGFR are overexpressed in the intrinsic Olaparib-resistant Rv1 cell line (A) The cell growth of Rv1 cells versus LNCaP and C4-2B cells treated with increasing doses of Olaparib for 72 h demonstrate robust resistance to Olaparib was determined by CCK-8. (B) Relative mRNA expression levels of IGFBP3 in Rv1 cells were determined by qPCR. (C) The protein expression of IGFBP3, Phospho-EGF receptor (Tyr1068), and γH2AX in Rv1 cells was determined by western blot analysis. Tubulin served as a loading control. (D) The protein expression of phospho-DNA-PKcs (Ser2056) in Rv1 cells was determined by western blot analysis. Tubulin served as a loading control. (E) The IC50 value of Rv1 cells treated with Gefitinib via cell growth were determined by CCK-8. (F) The cell growth of Rv1 treated with 10 μM Olaparib and 10 μM Gefitinib for 72 h was determined by CCK-8. The combination drug index (CDI) of Olaparib and Gefitinib in Rv1 was calculated. p value less than 0.05 was considered significant (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001). Statistical analysis was performed using Student’s t test.

Article Snippet: Phospho-DNA-PKcs (Ser2056) , Cell Signaling Technology , Cat#: 68716, RRID: AB_2939025.

Techniques: CCK-8 Assay, Expressing, Western Blot, Control

Journal: iScience

Article Title: IGFBP3 promotes resistance to Olaparib via modulating EGFR signaling in advanced prostate cancer

doi: 10.1016/j.isci.2024.108984

Figure Lengend Snippet:

Article Snippet: Phospho-DNA-PKcs (Ser2056) , Cell Signaling Technology , Cat#: 68716, RRID: AB_2939025.

Techniques: Recombinant, Negative Control, Transfection, Reverse Transcription, Protease Inhibitor, Western Blot, Saline, CCK-8 Assay, Viability Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Software

Journal: Cell reports

Article Title: Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by PrimPol

doi: 10.1016/j.celrep.2025.115360

Figure Lengend Snippet: (A–C) Proximity ligation assay showing the specific colocalization of Polκ or Polη with BPDE adducts in wild-type and BRCA2-knockout (BRCA2 KO ) (B and C) HeLa cells. Single antibody controls are used to confirm the specificity of the PLA signals observed. Quantifications (A and B) and representative micrographs (C), with scale bars representing 10 μm, are shown. At least 75 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). (D–G) SIRF experiments showing the specific recruitment of Polκ to nascent DNA upon treatment with BPDE in HeLa cells. Knockdown of Polκ is used as control to confirm the specificity of the SIRF signals observed. Quantifications (D–F) and representative micrographs (G), with scale bars representing 10 μm, are shown. At least 75 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Western blots confirming Polκ knockdown are shown in . (H–J) SIRF experiments showing the recruitment of Polη to nascent DNA upon treatment with BPDE or cisplatin in HeLa cells. Knockdown of Polη is used as control to confirm the specificity of the SIRF signals observed. Quantifications (H and I) and representative micrographs (J), with scale bars representing 10 μm, are shown. At least 75 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Western blots confirming Polη knockdown are shown in . See also and .

Article Snippet: Antibodies used were: Polκ (Bethyl A301–977A), Polη (Cell Signaling Technology 13848) and BPDE-DNA (Santa Cruz Biotechnology sc-52624).

Techniques: Proximity Ligation Assay, Knock-Out, Two Tailed Test, Knockdown, Control, Western Blot

Journal: Cell reports

Article Title: Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by PrimPol

doi: 10.1016/j.celrep.2025.115360

Figure Lengend Snippet: (A) SIRF assays showing the recruitment of PrimPol to nascent DNA upon treatment with BPDE or cisplatin in HeLa cells. Knockdown of PrimPol is used as control to confirm the specificity of the SIRF signals observed. At least 80 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). A schematic representation of the assay conditions is shown at the top. (B–E) SIRF assays showing that depletion of PrimPol suppresses the recruitment of TLS polymerases Polκ (B and D) and Polη (C and E) to nascent DNA upon adduct formation in HeLa cells. At least 75 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Western blots confirming PrimPol knockdown are shown in . (F–H) SIRF experiments showing that PCNA ubiquitination on nascent DNA upon adduct induction requires PrimPol in HeLa cells. Quantifications (F and H) and representative micrographs (G), with scale bars representing 10 μm, are shown. Knockdowns of RAD18 and USP1 were used as controls. At least 75 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Western blots confirming RAD18 and USP1 knockdowns are shown in and . See also – and .

Article Snippet: Antibodies used were: Polκ (Bethyl A301–977A), Polη (Cell Signaling Technology 13848) and BPDE-DNA (Santa Cruz Biotechnology sc-52624).

Techniques: Knockdown, Control, Two Tailed Test, Western Blot, Ubiquitin Proteomics

Journal: Cell reports

Article Title: Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by PrimPol

doi: 10.1016/j.celrep.2025.115360

Figure Lengend Snippet: (A–F) S1 nuclease DNA fiber combing assays showing that Polκ knockdown specifically increases nascent strand ssDNA gap formation upon BPDE exposure (A–C) and Polη knockdown specifically increases nascent strand ssDNA gap formation upon cisplatin exposure (D–F) in HeLa cells. REV1 depletion increases nascent strand ssDNA gaps upon both BPDE and cisplatin exposure (A, C, D, and F). (A and D) The ratio of CldU to IdU tract lengths from a representative experiment is presented, with the median values marked on the graphs and listed at the top. At least 55 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann-Whitney, two-tailed). (B, C, E, and F) Quantification of median CldU/IdU ratios from three independent experiments. The mean values and standard deviations are shown. Asterisks indicate statistical significance (t test, two-tailed, unpaired). The data for the other two experiments are presented in . Schematic representations of the assay conditions are shown at the top. (G–I) SIRF experiments showing that the recruitment of nucleases MRE11 (G) and EXO1 (H and I) to nascent DNA upon BPDE exposure in HeLa cells is increased by Polκ knockdown. At least 68 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance (t test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. (J and K) S1 nuclease DNA fiber combing assay showing that EXO1 deletion suppresses ssDNA gap accumulation in Polκ-depleted HeLa cells. (J) The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graphs and listed at the top. At least 65 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann-Whitney, two-tailed). (K) Quantification of median CldU/IdU ratios from three independent experiments. The mean values and standard deviations are shown. Asterisks indicate statistical significance (t test, two-tailed, unpaired). The data for the other two experiments are presented in . Schematic representations of the assay conditions are shown at the top. See also and .

Article Snippet: Antibodies used were: Polκ (Bethyl A301–977A), Polη (Cell Signaling Technology 13848) and BPDE-DNA (Santa Cruz Biotechnology sc-52624).

Techniques: Knockdown, MANN-WHITNEY, Two Tailed Test