Journal: Molecular cell

Article Title: REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps

doi: 10.1016/j.molcel.2021.08.016

Figure Lengend Snippet: A) Representative image of a replication fork detected by EM in BRCA1 -mutant HCC1937 cells. ssDNA gaps at the fork junction and internal gaps are indicated by brown and green arrows, respectively. B) Fold change in the percentage of replication forks with the indicated number of internal ssDNA gaps in HCC1937 cells relative to BRCA1-reconstituted HCC1937 cells. Values of individual experiments are indicated as dots. Columns represent the mean ± SEM of independent biological replicates (n = 3). Statistical analysis was conducted by unpaired t-test (*p < 0.05). C) BRCA1, RAD18, PCNA and ubiquitinated PCNA (PCNA-Ub) levels in WT and RAD18 KO (#1, #2) U2OS cells transfected with the indicated siRNAs, as detected by western blot. Vinculin is shown as control. D) Viability of WT and RAD18 KO (#1, #2) U2OS cells at day 5 and 10 after treatment with control or BRCA1 siRNA. Cell viability is represented as a percentage of viable BRCA1-depleted cells relative to control siRNA-treated cells. Values of individual experiments are indicated as dots. Columns represent the mean ± SEM of independent biological replicates (n ≥ 3). Statistical analysis was conducted by unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001). E) BRCA2 and RAD18 levels in WT and RAD18 KO (#1, #2) U2OS cells transfected with the indicated siRNAs, as detected by western blot. Vinculin is shown as control. F and H) Viability of WT and RAD18 KO (#1, #2) U2OS cells after treatment with the indicated siRNAs. Graphical representation and statistical analysis were conducted as in (D) (n = 3). G) Percentage of apoptotic cells determined by propidium iodide/Annexin V staining and flow cytometry after transfection of the indicated siRNAs. Graphical representation and statistical analysis were conducted as in (D) (n = 3).

Article Snippet: BRCA1-deficient and BRCA1-reconstituted MDA-MB-436 cells (2 × 10 6 ) were injected subcutaneously into flanks of female, 6–8 week-old NU/J (nude mice from Jackson laboratories) mice.

Techniques: Mutagenesis, Transfection, Western Blot, Control, Staining, Flow Cytometry

Journal: Molecular cell

Article Title: REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps

doi: 10.1016/j.molcel.2021.08.016

Figure Lengend Snippet: A) Viability of WT, REV1 KO, RAD18 KO and REV1/RAD18 KO U2OS cells after treatment with the indicated siRNAs. Cell viability is represented as a percentage of viable cells relative to control siRNA-treated cells. Values of individual experiments are indicated as dots. Columns represent the mean ± SEM of independent biological replicates (n = 3). Statistical analysis was conducted by unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). B) Viability of UWB1.289 and MDA-MB-436 cells with and without BRCA1 re-expression after transfection of Cas9 protein in complex with the indicated sgRNAs. Cell viability is represented as a percentage of viable cells relative to control sgRNA-treated cells (n = 4). Graphical representation and statistical analysis were conducted as in (A). C-D) Schematic of the IdU/CldU pulse-labeling protocol with S1 nuclease treatment (top). Dot plot and median of CldU tract lengths (μm) in PRIMPOL KO cells complemented or not with PRIMPOL WT or mutant cDNAs and subjected to the indicated experimental conditions (bottom). Doxycycline (DOX) induces the expression of Cas9 in cells carrying Cas9 and RAD18 sgRNA constructs. P-values were calculated by Mann-Whitney test (****p < 0.0001). E) Viability of WT and PRIMPOL KO U2OS cells transfected with the indicated sgRNAs in complex with Cas9 after treatment with the indicated siRNAs. Cell viability is represented as a percentage of viable cells relative to control siRNA-treated cells (n = 4). Graphical representation and statistical analysis were conducted as in (A). F) Viability of PRIMPOL KO U2OS cells with and without complementation with PRIMPOL WT or mutant cDNAs after treatments with the indicated siRNAs and DOX, as in (C) and (D). Graphical representation and statistical analysis were conducted as in (A).

Article Snippet: BRCA1-deficient and BRCA1-reconstituted MDA-MB-436 cells (2 × 10 6 ) were injected subcutaneously into flanks of female, 6–8 week-old NU/J (nude mice from Jackson laboratories) mice.

Techniques: Control, Expressing, Transfection, Labeling, Mutagenesis, Construct, MANN-WHITNEY

Journal: Molecular cell

Article Title: REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps

doi: 10.1016/j.molcel.2021.08.016

Figure Lengend Snippet: A) Survival analysis in the indicated breast and ovarian cell lines following treatment with JH-RE-06. Cell survival is expressed as percentage relative to the untreated control, and data represent the mean ± SEM of three replicates per condition. B) EC50 of JH-RE-06 per cell line obtained from the survival curves shown in (A) (left panel). Grouped analysis of JH-RE-06 EC50 values in BRCA1-deficient and BRCA1-proficient cells (right panel). Statistical analysis was conducted by unpaired t-test (*p < 0.05). C-E) Survival analyses upon JH-RE-06 treatment of the indicated BRCA1-deficient cell lines with or without BRCA1 re-expression. Data analysis and graphical representation were conducted as in (A). Statistical analysis was performed by unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001). F-G) Viability of the indicated BRCA1-deficient and BRCA1-reconstituted cell lines upon treatment with olaparib, cisplatin, JH-RE-06 and combinations of olaparib or cisplatin with JH-RE-06 at the indicated doses. Cell survival is expressed as percentage relative to the untreated control, and data represent the mean ± SEM of three replicates per condition. Statistical analysis was conducted by unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: BRCA1-deficient and BRCA1-reconstituted MDA-MB-436 cells (2 × 10 6 ) were injected subcutaneously into flanks of female, 6–8 week-old NU/J (nude mice from Jackson laboratories) mice.

Techniques: Control, Expressing

Journal: Molecular cell

Article Title: REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps

doi: 10.1016/j.molcel.2021.08.016

Figure Lengend Snippet: A-B) Viability of HCC1937 (A) and UWB1.289 (B) cells treated with the indicated siRNAs and JH-RE-06 (2.5 μM and 1.5 μM, respectively). Cell viability is expressed as percentage relative to the untreated control. Values of individual experiments are indicated as dots. Columns represent the mean ± SEM of independent biological replicates (n = 3). Statistical analysis was conducted by unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). C) Viability of MDA-MB-436 cells treated with control and SMUG1 siRNAs at the indicated JH-RE-06 doses. Graphical representation and statistical analyses were conducted as in (A) and (B). D) Viability of HCC1937 cells treated with control, SMUG1 and PRIMPOL sgRNAs at the indicated JH-RE-06 doses. Graphical representation and statistical analyses were conducted as in (A) and (B). E) PRIMPOL and SMUG1 levels in HCC1937 following transfection of the indicated siRNAs, as detected by western blot. Tubulin is shown as control. F) Viability of HCC1937 cells following treatment with the indicated siRNAs and doses of JH-RE-06. Graphical representation and statistical analysis were conducted as in (A) and (B). G) Schematic of the IdU/CldU pulse-labeling protocol with JH-RE-06 treatment followed by S1 nuclease digestion (top). Dot plot and median of CldU tract lengths (μm) in HCC1937 cells subjected to the indicated experimental conditions (bottom). P-values were calculated by Mann-Whitney test (****p < 0.0001).

Article Snippet: BRCA1-deficient and BRCA1-reconstituted MDA-MB-436 cells (2 × 10 6 ) were injected subcutaneously into flanks of female, 6–8 week-old NU/J (nude mice from Jackson laboratories) mice.

Techniques: Mutagenesis, Control, Transfection, Western Blot, Labeling, MANN-WHITNEY

Journal: Molecular cell

Article Title: REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps

doi: 10.1016/j.molcel.2021.08.016

Figure Lengend Snippet: A) Representative images of organoids of BRCA1-deficient and BRCA1-reconstituted UWB1.289 cells treated with DMSO or JH-RE-06 (1 μM) for 20 days. Scale bar = 400 μm. B) Number of UWB1.289 organoids per well treated as in (A). Values of individual experiments are indicated as dots. Columns represent the mean ± SEM of independent biological replicates (n = 3). Statistical analysis was conducted by unpaired t-test (**p < 0.01). C and E) Growth curves of xenograft tumors induced by MDA-MB-436 cells with or without BRCA1 re-expression upon treatment with vehicle control or JH-RE-06. Error bars represent SEM of 11 and 5 xenograft tumors per treatment group in (C) and (E), respectively. P-values of tumor volume differences between groups were calculated by unpaired t-test (*p < 0.05). D and F) Representative images (top panel) and dot plot with mean (bottom panel) of xenograft tumor masses collected at day 46 of the experiments shown in (C) and (E). The reference ruler is in cm. Statistical analysis between treatment groups was conducted using unpaired t-test (*p < 0.05).

Article Snippet: BRCA1-deficient and BRCA1-reconstituted MDA-MB-436 cells (2 × 10 6 ) were injected subcutaneously into flanks of female, 6–8 week-old NU/J (nude mice from Jackson laboratories) mice.

Techniques: Expressing, Control