Additional file 1 of Inhibition of autophagy by chloroquine prevents resistance to PI3K/AKT inhibitors and potentiates their antitumor effect in combination with paclitaxel in triple negative breast cancer models
Creators: name: Cocco, Stefania
name: Leone, Alessandra
name: Roca, Maria Serena
name: Lombardi, Rita
name: Piezzo, Michela
name: Caputo, Roberta
name: Ciardiello, Chiara
name: Costantini, Susan
name: Bruzzese, Francesca
name: Sisalli, Maria José
name: Budillon, Alfredo
name: De Laurentiis, Michelino
Publisher: name: figshare
Year: 2022
Subjects: subject: Medicine
subject: Molecular Biology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Cancer
subject: Hematology
subject: Plant Biology
subject: FOS: Biological sciences

description: Additional file 1. Fig. S1. Ipatasertib and taselisib determines antitumor effects in MDAMB361 cells. a Ipatasertib and taselisib reduce cell proliferation and ability to prevent clonogenic formation after daily administration for 10 days of IC30 doses for each drug or at fixed drug dose of 1µM, expressed as % of CTL. Each experiment is representative of three independent experiments. b Ipatasertib and taselisib treatment impair 3D tumor spheroid derived from MDAMB361 breast cancer cells. Representative images of first generation 3D tumor spheroid, exposed to ipatasertib and taselisib, administrated to IC30 doses for 72h. Tumor cell growth was reduced. Quantification of ATP was used to measure reduction of cellular growth, expressed as % of CTL. Each experiment is representative of three independent experiments. Statistically significant results are reported (*** indicates P &lt; 0.0005, ** indicates P &lt; 0.005 and * indicates P &lt; 0.05). c The exposure to fixed dose (IC50) of ipatasertib and taselisib induces the reduction of expression of phospho-mTOR, associated with the increase of autophagy signaling, as showed by increase of LC3 II/LC3 I ratio by immunoblot assay by reduction of p62 and increase of ATG5 after 24h of exposure in MDAMB361 cells (*** indicates P &lt; 0.0005, ** indicates P &lt; 0.005 and * indicates P &lt; 0.05). d The addition of CQ 10µM to ipatasertib and taselisib (IC50) induces accumulation of LC3II and p62 protein expression after 24h, due the reduction of autophagic flux, while expression of cleaved parp was not significant increased in taselisib +CQ and in ipatasertib+CQ groups in MDAMB361 cell line. e Representative confocal images of MDAMB361 cell lines immuno-stained with anti-LC3IIb antibody reveals accumulation of autophagosomes (green dots) in treatments with ipatasertib, taselisib, CQ or combinations, due to the induction of autophagy or reduction of autophagic flux exerted by CQ. (*** indicates P &lt; 0.0005, ** indicates P &lt; 0.005 and * indicates P &lt; 0.05). Fig. S2. Ipatasertib and taselisib induce cell cycle alteration as well as apoptosis in Breast cancer cells. a The reduction in proliferation was also associated with perturbation in the cell cycle, after 24h of treatment with ipatasertib and taselisib at IC30 doses, leading to an increase in the G1/G2 phase depending on the cell lines (MCF7, SKBR3, MDAMB361 and MDAMB231) expressed as % of CTL (b) or apoptosis measured by enhancement of Annexin V-FITC and Propidium Iodide, after 24 of treatment at IC30 doses, expressed as % of CTL. Similar representative experiments have yielded similar results. Statistically significant results were reported (*** indicates P &lt; 0.0005, ** indicates P &lt; 0.005 and * indicates P &lt; 0.05). Fig. S3. Ipatasertib and taselisib induce autophagy in MDAMB231 cells. Representative confocal images of MDAMB231 cells overexpressing GFP-LC3 and immuno-stained with anti-p62 ab, reveal accumulation of autophagosomes (green dots) that colocalise with p62 (Merge) in CQ treatments, due to the reduction in autophagic flux exerted by CQ. Ipatasertib, taselisib and CQ were administered at a 10µM dose for 24h. Similar representative experiments yielded similar results. Statistically significant results were reported (*** indicates P &lt; 0.0005, ** indicates P &lt; 0.005 and * indicates P &lt; 0.05). Fig. S4. Chloroquine potentiate antitumor effects induced by ipatasertib and taselisib. a The addition of CQ 1-10-20µM to ipatasertib or taselisib inhibits cell proliferation measured by SRB assay after 48h in MCF7 and SKBR3, and 72h in MDAMB361 on treatments indicated, expressed as % of CTL. Each experiment is representative of three independent experiments. (b) The addition of CQ 10µM to ipatasertib and taselisib (IC30 for MCF7, SKBR3, MDAMB361 and 10µM for MDAMB231) causes an increase of cytotoxicity measured by LDH release after 24h, expressed as % of CTL. Each experiment is representative of three independent experiments. Statistically significant results were reported (*** indicates P &lt; 0.0005, ** indicates P &lt; 0.005 and * indicates P &lt; 0.05). Fig. S5. Taselisib induce anti-proliferative effects via autophagy mediated by ATG5. a ATG-5-siRNA transfection in MDAMB231 cell lines induces a relevant reduction in ATG5 protein expression compared with the control non-transfected or scramble transfected cells within 24 h from transfection b) In the SRB-based proliferation assay, TAS+ATG5-siRNA and TAS+ATG5-siRNA+paclitaxel groups showed a significant reduction in the proliferation rate after 72h of treatment. Ipatasertib and taselisib were administered at a 1µM dose and paclitaxel at a 0.5nM dose. Statistically significant results were reported (* indicates p &lt; 0.05).