hGLE1 Knockdown and Add-back Protocol

HeLa cells were cultured in complete DMEM media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Norcross, GA) at 37°C in 5% CO₂. Knockdown add-back of hGLE1 was performed using the previously validated protocol^{17 (link)}. Negative control siRNA and hGLE1 siRNAs were purchased from Qiagen (Valencia, CA). hNUP42 SMARTpool siRNA was purchased from Dharmacon (Lafayette, CO). Cells were reverse-transfected with indicated 25 nM siRNAs using HiPerFect (Qiagen) and then transfected 24 h later with relevant constructs (siRNA-resistant mCherry-hGLE1B^R or mCherry-hgle1B^R-QKED>AAAA for Figure 4; siRNA-resistant GFP-hGLE1B^R or GFP-hgle1B^R-KK>QQ for Figure 5) using Fugene 6 (Promega, Madison, WI) per manufacturer’s instructions.