Additional file 1 of LCK facilitates DNA damage repair by stabilizing RAD51 and BRCA1 in the nucleus of chemoresistant ovarian cancer
Creators: name: Dey, Goutam
name: Bharti, Rashmi
name: Braley, Chad
name: Alluri, Ravi
name: Esakov, Emily
name: Crean-Tate, Katie
name: McCrae, Keith
name: Joehlin-Price, Amy
name: Rose, Peter G.
name: Lathia, Justin
name: Gong, Zihua
name: Reizes, Ofer
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Plant Biology

description: Additional file 1: Supplementary Fig. S1. Transfection efficiency of Myc tagged LCK in SKOV3 and CP70. SKOV3 and CP70 cells were transduced with EV or Myc tagged LCK plasmid by using lentiviral particle. Then, cells were checked for Myc and LCK expression. Supplementary Fig. S2. CP70 EV and CP70 LCK OE cells were treated with cycloheximide in a time dependent manner. Then, immunoblot analysis was performed to evaluate the expression of RAD51 and BRCA1 proteins (Main Fig. 1G, H). Half-lives were determined from digitized images. Supplementary Fig. S3. (A) CP70 WT, LCK KO (CRISPR/Cas9) and LCK OE (In CRISPR background) cells were treated with DMSO/etoposide 10µM for 24h. Then cells were kept in drug free media for another 24h. Then immunofluorescence study was performed to visualize γH2AX foci formation in different groups. (B) CP70 WT, LCK KO (CRISPR/Cas9) and LCK OE (In CRISPR background) cells were treated with DMSO/etoposide 10µM for 24h. Cells were put in drug free media for another 24h. Then immunofluorescence study was performed to visualize RAD51 foci formation in different groups. Supplementary Fig. S4. (A) CP70 cells (LCK OE, LCK Y394F, LCK K273R, and LCK Y192F, all constructs were introduced into the CP70 LCK KO cells) were treated with etoposide for 24h. Cells were then kept in drug free media for 24h. Immunofluorescence study was performed to visualize γH2AX foci formation. (B) CP70 cells (LCK OE, LCK Y394F, LCK K273R, and LCK Y192F) were treated with etoposide for 24h. Cells were then kept in drug free media for 24h. Immunofluorescence study was performed to visualize RAD51 foci formation. Supplementary Fig. S5. CP70 cells (LCK, LCK Y394F, LCK K273R, and LCK Y192F in LCK knock out background) were grown on cover slips and treated with etoposide for 24h followed by incubation for 0, 2, 4, 8 and 24h. Cells were then subjected to immunofluorescence analysis to visualize H2AX foci formation. Supplementary Fig. S6. (A) SKOV3 cells were treated with etoposide, and MMS for 24. After that cells were put in 24h in drug free media. Cells were then subjected to western blot analysis for checking protein expression. (B) DNA damage by UV radiation upregulates LCK phosphorylation. CP70 cells were treated with UV radiation for 1min, 2min and 4min. Cells were kept in serum enriched media for 24h. Then cells were subjected to western blot analysis to check the expression of P-LCK, T-LCK and BRCA1 expression. (C) SKOV3 LCK OE cells were treated with etoposide for 24h. Cells were then put in drug free media for another 24h. Cells were collected, and cytoplasmic and nuclear proteins were extracted for western blot analysis. Supplementary Fig. S7. (A, B) Protein quantification of T-LCK, BRCA1, and γH2AX in CP70 cells treated with etoposide and MMS (Main Fig. 3A). (C) Quantification of P-LCK and T-LCK in CP70 LCK OE cells treated with etoposide (Main Fig. 3B). (D) Quantification of BRCA1 protein in CP70 shCon, LCK KD1 and LCK KD2 cells treated with/without etoposide (Main Fig. 3E). (E) Quantification of BRCA1 protein expression in CP70 WT and LCK KO cells treated with/without etoposide (Main Fig. 3F). One way ANOVA analysis was performed with Tukey's multiple comparisons test to compare different groups (* p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001). Supplementary Fig. S8. LCK modulates expression of HR repair proteins. (A) Western blot of CP70 and SKOV3 cells containing various lentiviral EV, LCK KD, KO, and OE to determine effects on LCK, BRCA1, BRCA2 and RAD51 expression. We detected a significant basal level of LCK in WT cells. In EV-OE panel, we used empty vector and LCK OE plasmid transduced in LCK knock out cells. (B) Western blot analysis of CP70 and SKOV3 LCK OE cells treated with PP2 in a dose dependent manner for 48hrs, demonstrating the effects of a pharmacological inhibitor of LCK on P-LCK, T-LCK, BRCA1, BRCA2, RAD51 and γH2AX protein expression. Supplementary Fig. S9. Cell cycle analysis by flow cytometry. (A) CP70 cells (LCK OE and KO) were grown to 70% confluency. Cells were harvested by trypsinization and fixed with chilled ethanol. Cell were then stained with propidium iodide and subjected to flow cytometry. Experiments were replicated three times. (B) Percentage of cell population in each phase of cell cycle are presented. One way ANOVA analysis was performed with Tukey's multiple comparisons test to compare different groups (* p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001). Supplementary Fig. S10. (A) Pharmacological inhibition of LCK attenuates HR repair proteins in ovarian cancer cells. CP70, SKOV3 and CRL1978 cells were treated with the LCKi, PP2 for 48h. Cells were harvested, lysed, and analyzed by immunoblot to assess protein expression of BRCA1, BRCA2, RAD51, and γH2AX. (B) Western blot analysis in different cells to check the expression of LCK and BRCA1. U2OS is osteosarcoma cell line which was used in DR GFP assay. U2OS and U2OS: DR-GFP cells were examined for checking LCK and BRCA1 expression. These cells were also found to express LCK and BRCA1 like CP70 and SKOV3 cells. (C) Representative image of flow cytometry from DRGFP assay. Upper panel is shows GFP population of U2OS cells treated with PP2 and lower panel is shows GFP population in Sh Control, LCK KD1 and KD2 groups of U2OS cells. (D) CRL1978 cells were treated with increasing concentrations of PP2 for 48h and cells were harvested, lysed, and immunoblotted for Ku70, and Ku80 protein expression. GAPDH was used as loading control. Supplementary Fig. S11. (A) CP70 Parental cells and CP70 LCK KO and CP70 CD55 OE (In KO background) cells were treated with Olaparib in dose dependent manner for 12 days. After that colonies were stained with crystal violet and images were captured. (B) Number of Colony formation was counted and plotted as percentage of colony formation in the graph (Main fig 6G). IC50 values were shown in the table. Supplementary Fig. S12. (A, B) CP70 Sh Con or LCK knock down cells were treated with Olaparib in a dose dependent manner for 12 days. Number of colonies was counted and plotted using graph pad prism. (C, D) SKOV3 Sh Con or LCK knock down cells were treated with Olaparib in dose dependent manner for 12 days. Number of Colony formation was counted and plotted in the graph. Supplementary Fig. S13. (A) TUNEL assay to detect DNA fragmentation in tumor tissue sections. TUNEL positive cells were counted from five images and plotted in graph (Main fig. 8D). (B) IHC staining of γH2AX of tumor sections from different groups. γH2AX positive cells were counted from five images and plotted in graph (Main fig. 8E). (C) CD31 expression (Indicator of microvessel density and growth) of tumor sections from different group of mice. Microvessel density was counted from five images and plotted in graph (Main fig. 8F). Images are representative of two tumors from each cohort. We quantified the staining from 5 fields from each mouse. Images were captured at 20X magnification. One way ANOVA analysis was performed with Tukey's multiple comparisons test to compare different groups (* p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001).