Experimental readouts

Quantitative PCR analysis (qPCR) involved RNA isolation with TRIzolTM by manufacturer instructions (Invitrogen; Carlsbad, CA, USA). cDNA synthesis used Oligo(dT)20 Primer (Invitrogen), SuperScript IV Reverse Transcriptase (including buffer + DTT) (Invitrogen), dNTPs (Genecraft; Lüdinghausen, Germany) and Random Hexamers dN6 (Thermo Fischer). qPCR used Luminoct® SYBR Green qPCR Ready Mix (Sigma-Aldrich) and the following primer pairs (IDT; Coralville, Iowa, USA and Eurofins (Luxembourg):

ALDH1A1 Fw: GCACGCCAGACTTACCTGTC, Rv: CCTCCTCAGTTGCAGGATTAAAG; KDM1A Fw: TGACCGGATGACTTCTCAAGA, Rv: GTTGGAGAGTAGCCTCAAATGTC; KDM1B Fw: CTCTCCTGTGGGGAACATTTC, Rv: GACTAGGTTCGGTTTTGCCATT; KDM2B Fw: GGGTTCCCCTGATATTTCGAGA, Rv: GCTCCCCACTAGGAGTTTGAC; KDM5A Fw: GTCACCTGGAGCTAAGGCAC, Rv: CCGTTTCCGTTTCTTCTCTG; KDM5B Fw: AGTGGGCTCACATATCAGAGG, Rv: CAAACACCTTAGGCTGTCTCC; KDM5C Fw: CTTGCTACGCTCCCACTACG, Rv: TGTGTTACACTGCACAAGGTTG; KDM5D Fw: CAAGACCCGCTTGGCTACATT, Rv: TTGGACGCGAGGAGTAAATCT; KDM6A Fw: TACAGGCTCAGTTGTGTAACCT, Rv: CTGCGGGAATTGGTAGGCTC; RPL37A Fw: GACGTACAATACCACTTCCGC, Rv: GGAGCGTCTACTGGTCTTTCA.

A PCR Mastercycler nexus GX2 (Eppendorf, Hamburg, Germany) and a CFX96 Touch real-time cycler (BioRad, Hercules, CA, USA) were used. Relative gene expression was calculated by normalization to the housekeeper gene RPL37A.

Flow cytometry was conducted per standard methodology on a FACS Celesta (BD Biosciences; San Jose, CA, USA) using the FACS Diva software version v 8.0.1.1 (BD Biosciences). Data analysis was performed using FlowJo software, version v10.5.3. The gating strategy for detection of KDM5B^high cells, included the setting of a threshold on the 5% highest fluorescence intensity (TOP 5% EGFP-KDM5B reporter) as determined in the respective control sample (naive cells or DMSO). The control gate was projected onto each further sample to determine the frequency of experimental cells reaching the threshold.

The 7-AAD Viability Staining Solution (0.25μg/1x10⁶ cells; Thermo Fischer) was applied to single cell suspensions of indicated samples, derived from drug exposure studies, 5-10 minutes prior to flow cytometry analysis.

CellTrace™ Far Red solution (5μM in PBS; Thermo Fischer #{"type":"entrez-nucleotide","attrs":{"text":"C34572","term_id":"2370713","term_text":"C34572"}}C34572) was applied by manufacturer’s instruction on indicated cell samples under adherent proliferative conditions, one hour before the indicated drug exposure. Proliferation analysis by flow cytometry was performed five days later.

Quantification of cellular phenotypes was performed on 0.25-0.5x10⁶ of the indicated patient cells in FACS buffer (2mM EDTA, 0.1 % BSA in 1x PBS) with human Fc Block (1:100; BD Biosciences, Franklin Lakes, NJ, USA, #564220, RRID: AB_2869554) for 15 min at RT. Cells were fixed and permeabilized by the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences) according to the manufacturer’s protocol. Conjugated primary antibodies included: ALDH1A1 (FITC, 1:5, Abcam #ab275646, RRID: N/A) and pAKT (Ser473) (Alexa488, 1:2.5, BD Biosciences #560404, RRID: AB_1645342). The unconjugated KDM5B antibody (rabbit, 1:25, Novus #NB100-97821, RRID: AB_1291176) was combined with an Alexa-Fluor 555 coupled secondary antibody (goat anti-rabbit IgG, 1:200, Thermo Fisher Scientific #A21428, RRID: AB_2535849) or alternatively with an Alexa-Fluor 647 coupled secondary antibody (donkey anti-rabbit IgG, 1:400, BioLegend #406414; RRID:AB_2563202). Antibody solutions were incubated with cells for 30 min on ice. Prior to flow cytometry analysis, cells were washed and resuspended in FACS buffer.

The caspase assay was evaluated for active Caspase-3 accordingly, using appropriate antibodies (FITC, 1:5, BD Bioscience #559341, RRID: AB_397234) and methods prior to flow cytometry.

Cell confluence was determined in the cellular assays at indicated time points longitudinally using software-based cell recognition (Nyone®, Synentec, Elmshorn, Germany).

Cell viability was evaluated via alamarBlueTM assay (Life Technologies) following the manufacturer’s instructions. Readout was performed on a Tecan Infinite F200 instrument (Tecan, Männedorf, Switzerland).

Protein expression analysis involved scrapping of cells using a lysis buffer (Pierce™ RIPA Buffer (Life Technologies) containing cOmplete™ Protease Inhibitor Cocktail (Roche; Basel, Switzerland) and PhosSTOP™ (Sigma-Aldrich). Protein concentration was measured using Thermo Scientific Pierce BCA Protein Assay (Thermo Fischer) and a Tecan Infinite F200 instrument. Total protein was separated by SDS-PAGE (4–15% Mini-PROTEAN® TGX™ Precast Protein Gels; BioRad) and transferred onto nitrocellulose membranes (Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs; Biorad) by electroblotting. Membranes were blocked in 5% milk powder (Carl Roth GmbH, Karlsruhe, Germany) in PBS (Thermo Fischer) with 0.5% TWEEN®-20 (Sigma-Aldrich) (PBST) or alternatively in TBS (TRIS-Hydrochlorid (Carl Roth); Sodium chloride (Carl Roth)) with 0.5% TWEEN®-20 (Sigma-Aldrich) (TBST) for 1 hour. The membranes were supplied with primary antibodies (KDM5B (1:250, Sigma-Aldrich #HPA027179, RRID: AB_1851987); PTEN XP® (1:1000, Cell signaling #9188P, RRID: AB_2253290); phospho-AKT (Ser473) XP® (1:2000, Cell signaling #4060S, RRID: AB_2315049); phospho-GSK3ß (Ser9) (1:1000, Cell signaling #5558P, RRID: AB_10013750); phospho-mTOR (Ser2448) XP® (1:1000, Cell signaling #5536P, RRID: AB_10691552); Cyclin D3 (1:1000, Cell signaling #2936, RRID: AB_2070801)) in 5% milk powder in PBST overnight at 4°C or for 1 hour at room temperature (β-actin (1:5000, Sigma-Aldrich #A5441, RRID: AB_476744). The primary antibodies PI3 Kinase p85 1:1000, Cell signaling #4292, RRID: AB_329869) and PI3 Kinase p110α (1:1000, Cell signaling #4255, RRID: AB_659888) were supplied in 5% BSA (Carl Roth) in TBST overnight at 4°C. The membranes were detected with horseradish peroxidase (HRP)-coupled secondary antibodies (anti-rabbit IgG, HRP-linked (1:2000, Cell signaling #7074, RRID: AB_2099233); Anti-mouse IgG, HRP-linked (1:2000, Cell signaling #7076, RRID: AB_330924)) using Clarity™ Western ECL Substrate (Biorad) and the ChemiDoc System (Biorad). For stripping the membrane was incubated with suitable stripping buffer before blocking with 5% milk and staining with primary and secondary antibodies as described above. Intensity quantification and normalization to ß-actin was performed using Image Lab™ software version 6.0.1 (Biorad).