Fig. 3: Additional validation of HORMAD1-driven POLH dependency.

A Bar plot displaying reduced surviving fractions of clonally-derived HORMAD1-inducible SUM159 cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) transfected with an siRNA pool or 4 individual siRNAs targeting POLH and exposed to HORMAD1 expression for 14 days (in total). Non-targeting (siALLSTAR) and targeting (siPLK1) siRNAs were used as transfection controls and surviving fractions calculated from mock-transfected cells. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t tests (***p = < 0.0001, **p = < 0.001, *p = < 0.05). B Bar plot displaying the percentage of POLH mRNA expression following siRNA-mediated depletion of POLH described in A, measured by RT-qPCR and normalised to ACTB. C Bar plot displaying reduced surviving fractions of clonally-derived HORMAD1-inducible SUM159 cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) expressing constitutive Cas9-mCherry, transfected with 5 Edit-R crRNAs targeting POLH, and exposed to HORMAD1 expression for 14 days (in total). Surviving fractions were calculated relative to Cas9-expressing mock-transfected controls. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t-tests (***p = < 0.0001, **p = < 0.001, *p = < 0.05). D Western blot analysis of POLH protein knockout from experiment C. E, F Left, growth curves displaying reduced cellular growth of HORMAD1-expressing breast cancer cell lines E HCC38 and F BT549 expressing constitutive Cas9-mCherry and bulk-transfected with 3 POLH-targeting Edit-R crRNAs. Cell number was normalised relative to T0 counts. Error bars indicate SD from mean effects (n = 3). p-values represent two-way repeated-measures ANOVA. Right, western blot analysis of HORMAD1 expression and POLH protein knockout from experiments described in left panel.