Ca2 free dmem

mIMCD3 cells grown on 65mm Transwell filters (Corning) for 7 days were subjected to Ca²⁺ switch as described in Straight et al.^{52 (link)}. Briefly, cells were washed with PBS-4mM EGTA and Ca²⁺ free DMEM (Life Technologies) containing 4mM EGTA was added to the cells for 45 min to disrupt cell junctions. Cells were then washed twice with normal culture medium (DMEM/F12) and TER was determined using a Millicell-ERS volt–ohm meter (Millipore) immediately after the addition of normal growth medium and at the indicated time points. 6hrs after Ca²⁺ switch, cells were fixed with 4% PFA and processed as described above.

HeLa, U2OS, HEK293T, and MEFs were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO₂. DMEM/F12 medium was used for culturing nontransformed RPE cells. WT or AMPKα1^−/−AMPKα2^−/− double knockout (AMPKα-KO) MEFs were previously described (Shen et al., 2013 (link)). For the experiment shown in Figure 6e, HeLa cells were cultured in regular medium or in Ca²⁺-free DMEM (Gibco, 21068–028) supplemented with dialyzed FBS (Sigma, F0392). Plasmids were transfected into cells using TransIT-LT1 (Mirus, for U2OS and HEK293T) or Lipofectamine® 3000 (Life Technologies, for HeLa) according to the protocols of the manufacturers. siRNA transfection was done using TransIT-siQUEST transfection reagent (Mirus). For pretreatment with CC, STO-609, BAPTA, BAPTA-AM, or AZD7762, cells were incubated with the compounds for 30 min before the addition of HU or aphidicolin to induce replication stress. For UV treatment, cellular culture medium was replaced with PBS, and cells were then exposed to UV (254 nm) at 60 mJ/cm² using a Stratalinker® UV Crosslinker. Cells were then cultured in fresh medium for 4 h before imaging or protein sample preparation.