Pcdk9

Antibodies and sources: CDK9, pCDK9, RNAPII, phospho-RNAPII, TGM2, Cyclin B1, Cyclin E1, and NUP98 (Cell Signaling Technology); CDC37, SPT5, Stomatin, PLK1, Cyclin A1 (Santa Cruz Biotechnology); BRD4, Cyclin T1 and GFP (Abcam); Caspase-8 (Enzo Life Sciences); β-Actin, pCDK9, Vimentin, and Flag (Sigma-Aldrich).
Reagents and sources: CellTiter-Blue Cell Viability assay and Caspase-Glo 3/7 assay (Promega); BrdU kit (Roche); Thymidine, 5-ethynyl uridine (EU), Azide-fluor 488 (Sigma-Aldrich); AnnexinV and 7AAD (BD); [γ-32P] ATP (3000Ci/mmol, Amersham Pharmacia); Trail, FasL (Enzo Life Sciences); PLA assay kit (Olink Biosciences); BAY1251152, Cisplatin, and Carboplatin (Selleckchem); BioCoat Matrigel invasion chamber (Corning); Migration chamber (Ibidi); RNeasy Plus kit (Qiagen), active GST-CDK9/Cyclin K (SRP5012, Sigma-Aldrich).
The following vectors were used: pCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene); p3xFlag-CMV-7.1 (E7533, Sigma); pGEX-5X-3 (28–9545-55, GE Healthcare) and pEGFP-C2 (6083-1, Clontech). All siRNAs and primers were from Sigma-Aldrich.

Formalin-fixed, paraffin-embedded (FFPE) pre-treatment biopsies were subjected to an HRP technique (DAKO Envision Flex, Hamburg, Germany) [50 (link)] with Caspase-8 (Thermo Fisher Scientific) and pCDK9 (Sigma) antibodies at 1:150 and 1:50 dilutions, respectively. Next, dextran-polymer-conjugated horseradish peroxidase and 3,3′-diaminobenzidine (DAB) chromogen were used for visualization and hematoxylin solution for counterstaining. Negative control slides in the absence of primary antibodies were included for each staining procedure. Two investigators (F.R., I.K.), blinded to patient clinical information, performed the evaluation to minimize inter-observer variability. Marker expressions were dichotomized as “high” [Weighted Score (WS) > 6] or “low” (≤ 6) based on a combination of the fraction of positive cells [1 (0–25%), 2 (26–50%), 3 (51–75%) and 4 (> 75%)] and the staining intensities [1 + (weak), 2 + (moderate) and 3 + (intense)] [16 (link)]. Image acquisition was performed using an AxioScanZ1 slide scanner and Zen software (Zeiss, Jena, Germany).