Fig 2: Silencing of FANCI in cell lines with functional Fanconi Anemia pathway results in reduced expression of other FA complex components. (A) The effect of FANCI silencing on other key FA complex members, namely FANCA, FANCB, FANCC, FANCD2 and UBE2T (FANCT) on mRNA level in LNCaP cells analyzed using RT-qPCR. Bars represent mean±SD with n=3. P-values shown as asterisks (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001). (B) The effect of FANCI silencing on FANCI, FANCB, UBE2T and FANCD2 protein levels in LNCaP cells analyzed using immunoblotting. GAPDH antibody was used as a housekeeping control. White space was used to make explicit for the grouping of blots cropped from different gels. (C) Immunofluorescence images of LNCaP cells with either FANCI or control siRNA stained with γH2AX (red), which is a known marker for DNA damage. DAPI staining (blue) was used to visualize the nuclei of the cells. (D) Results of the immunofluorescence imaging of FANCI or control siRNA PC-3 cells stained with γH2AX (red) and nuclei DNA binding DAPI (blue). (E) The effect of Carboplatin (5 µM) on FANCI monoubiquitination in LNCaP prostate cancer cell line analyzed using Western blot of FANCI (140 kDa) and monoubiquitinated FANCI (150 kDa) in comparison to DMSO control cells. Upon Carboplatin exposure, a second band at about 150 kDa corresponding to monoubiquitinated FANCI is observed. White space was utilized to highlight the grouping of blots cropped from different gels. (F) Similarly, also in PC-3 cells exposure to Carboplatin results in monoubiquitination of FANCI based on Western blot analysis. (G) Immunofluorescence analysis of LNCaP cells exposed to either DMSO (control, ctrl) or Carboplatin (5 µM) using antibody against FANCI (red) to determine the localization and overall expression of FANCI. The formation of distinct nuclear foci in response to Carboplatin indicates activation of FANCI and FA pathway. (H) Immunofluorescence analysis of PC-3 cells exposed to DMSO or Carboplatin using FANCI antibody (red). Unlike in LNCaP cells, in PC-3 cells only faint FANCI signal was observed. In both LNCaP and PC-3 cells DAPI staining (blue) was used to visualize nuclear DNA.

Fig 3: TTFields increase DNA damage induced by carboplatin and PARPi, elevate levels of p21, and downregulate the FA-BRCA pathway. A2780 (A, D, G), OVCAR-3 (B, E, H), and A2780cis (C, F, I) human ovarian cancer cells (HRP, HRD, and platinum-resistant cells, respectively) were treated for 72 hr with carboplatin (A–C), olaparib (D–F), or niraparib (G–I), alone or together with TTFields (200 kHz, 1 V/cm RMS), followed by immunofluorescent detection of ɣH2AX foci formation. Drug concentrations: A2780 – 6 µM carboplatin, 1 µM olaparib, and 0.5 µM niraparib; OVCAR-3 – 16 µM carboplatin, 0.5 µM olaparib, and 0.8 µM niraparib; A2780cis – 36 µM carboplatin, 10 µM olaparib, and 1.5 µM niraparib. Representative images show staining with anti ɣH2AX antibody (green) and DAPI for nuclear visualization (blue) at x40 magnification; Scale bar, 20 µm. Values are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 relative to TTFields plus drug; One-way ANOVA, followed by Dunnett’s post hoc analysis. A2780 (J) and (L), OVCAR-3 (M), and A2780cis (K) and (N) human ovarian cancer cells were treated for 72 hr with TTFields (200 kHz, 1 V/cm RMS), followed by immunoblotting of cell lysates for expression of p21 (J, K), FANCB, FANCD2, FANCJ, and BRCA2 (L–N). Values are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 relative to control; Student’s T-tests.