T7402

LLC-PK1 cells (ATCC) were maintained in minimum essential medium (MEM) supplemented with 3% FBS. Mouse renal PTCs were developed as previously described (54 (link)). Cells were maintained at 33°C in DMEM/F-12 containing 2.5% FBS and IFN-γ and then transferred to 37°C for differentiation before use (54 (link)). The HEK293T (ATCC) cells were maintained in DMEM supplemented with 10% FBS. Cells were treated with AA (2.5–5 μg/mL; Sigma-Aldrich, A5512), PAC (10 μM; Sigma-Aldrich, T7402), or respective vehicles in complete medium for 48 hours. Primary PTCs were isolated from WT mice or CG1-KO mice as described previously (55 (link)). In brief, kidney cortex was minced, digested with collagenase (Worthington Biochemical Corporation) and trypsin inhibitor (Worthington Biochemical Corporation), and centrifuged in 32% Percoll medium to purify PTCs. Primary PTCs were then plated in collagen-coated dishes and maintained in DMEM/F-12 medium supplemented with insulin, transferrin, selenium, 0.05 μM hydrocortisone, and 50 μM vitamin C. To avoid changes in PTC phenotype due to repeated passages, these cells were plated as soon as they became confluent for experiments. Primary PTCs were treated with AA at a concentration of 5 to 10 μg/mL for the times indicated.

Purchased compounds included chemotherapeutics [paclitaxel (Sigma #T7402)], early stage autophagy inhibitor [PI-103 (LC Labs #P-9099)], late-stage autophagy inhibitors [bafilomycin A1 (Sigma #SML1661, LC Labs #B-1080 in LC3B puncta and autophagic flux assays, and Cayman Chemical #11038 for cytotoxicity assays)] and chloroquine (Sigma #C6628), and nucleocytoplasmic transport pathway inhibitors [importazole (Cayman Chemical #21491) and KPT-330 (Cayman Chemical #18127)] [52 (link), 58 (link)]. Structures of non-FDA-approved small molecules may be found here: bafilomycin A1 [59 ], importazole [60 (link)], and HTP-013 [12 (link)]. Photoaffinity labeled PHY analogs were created as previously published [17 (link), 61 (link)]. All compounds were suspended in dimethyl sulfoxide (DMSO), and the final vehicle concentration < 0.1% (v/v).

The human TNBC cell lines were cultured at 37 °C under 5% CO₂. MDA-MB-231 and HCC1806 cells were cultured in phenol red RPMI (Sigma-Aldrich, St. Quentin Fallavier, France), supplemented with 10% FBS (Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich). Hs578T cells were grown in DMEM medium containing phenol red (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich) and 10 µg/mL bovine insulin (Sigma-Aldrich). All cell lines were purchased from the ATCC (American Type Culture Collection). Cells were treated for 72 h with DMSO or Ethanol (vehicle) or different concentrations of 5-FU (F6627, Sigma-Aldrich), PTX (T7402, Sigma-Aldrich) and DOX (D1515, Sigma-Aldrich). All chemotherapeutic agents were dissolved in DMSO (5-FU), ethanol 100% (PTX) or sterile water (DOX) and were stored at −20 °C.