Journal: bioRxiv

Article Title: Molecular consequences of acute versus chronic CDK12 loss in prostate carcinoma nominates distinct therapeutic strategies

doi: 10.1101/2024.07.16.603734

Figure Lengend Snippet: (a) Stable CDK12(-) cells show fewer upregulated intronic APAs. Table with the number of APA sites down (DN), no change (NC), or up (UP) in isogenic paired models (CRISPR KO clones vs parental, or 189.4-CDK12 vs 189.4-vec). The ratio UP/DOWN indicates skew towards the IPA phenotype. (b) Validation of Tet-shRNA lines. Western blot with lysates from Tet-shRNA lines treated four days -/+ 100ng/mL doxycycline. (c) LuCaP189.4_CL cells are RAD51 competent. Irradiation and immunostaining (same as in ). Cells were exposed to 6Gy IR and fixed at 3h. Immunofluorescence staining was performed for γH2A.X and RAD51 and images were acquired by confocal microscopy. Left: representative images (white: DAPI, green: γH2A.X, purple: RAD51). Right: quantification of images (∼200-500 cells analyzed per treatment). Line is at mean and significance was determined by unpaired t-test (Mann-Whitney). (d-e) CDK12 knockdown does not prevent RAD51 foci. Additional immunostaining (same as in ) using LNCaP (d) and Skov3 (e) with Tet-shCDK12 or Tet-shBRCA2. Cells were treated four days -/+ dox. Graphs show mean with significance determined by one-way ANOVA (Kruskal-Wallis).

Article Snippet: Tet-inducible shRNA vectors were cloned as previously described( ) into EZ-Tet-pLKO-Hygro (Addgene 85972).

Techniques: CRISPR, Clone Assay, Biomarker Discovery, shRNA, Western Blot, Irradiation, Immunostaining, Immunofluorescence, Staining, Confocal Microscopy, MANN-WHITNEY, Knockdown