Fig 2: Validation of shRNA and siRNA-mediated knockdown of genes. (A) Four druggable putative synthetic lethal partners of GBF1: ARF1, PRKAA1, CSK, and HSP90; a putative GBF1 suppressor, ARF4; and a gene showing no GCA-induced chemogenomic interactions with GBF1, MSMO1, were silenced in HeLa cells via shRNA-mediated lentivirus transductions and selected on puromycin for 72 h. Cell pellets from each knockdown cell-line were collected, lysed, and the total protein concentrations were measured using a bicinchoninic acid assay. Equal amounts of total protein from the control and knockdown cells were resolved by SDS-PAGE, transferred to PVDF and probed with the indicated antibodies. (B) For experiments with siRNA, HeLa cells were transiently transfected with siRNAs targeting ARF1 and a nontargeting control. After 48 h post-transfection, the control and KD cells were harvested and replated for IF imaging experiment (Fig. 4, D–F, main text). Cell pellets were collected and used for WB analysis as described above. β-actin was used as a loading control. Protein depletion relative to the respective loading control was calculated using ImageJ software and the resulting percent knockdown efficiencies (% KD) are reported. Source data are available for this figure: SourceData FS2.

Fig 3: Validation of putative synthetic lethal interactions in HeLa cells.(A) ARF1 displays a robust synthetic lethal interaction with GBF1. ARF1, HSP90, CSK, PRKAA1, the control gene MSMO1, and the top GBF1 suppressor gene ARF4 were silenced in HeLa cells with shRNA-mediated lentivirus transductions and incubated with 1.5 or 4 µM golgicide A (GCA) or DMSO for 48 h. CellTiterBlue reagent was added and fluorescence measurements were collected. Live cells metabolize the reagent into fluorescent products and an increase in the fluorescence signal is directly proportional to the number of living cells. The percent viability at each GCA concentration was calculated by dividing the fluorescence from a GCA-treated sample by its matched DMSO-treated control. Changes in cell viabilities for each knockdown (KD) cell line were determined by comparing the respective percent viabilities to the MSMO1 KD control using a Brown Forsythe and Welch ANOVA multiple comparison test (Brown and Forsythe, 1974; Welch, 1951), with statistically significant differences are indicated as: * if p-value < 0.01; ** if p-value < 0.001; *** if p-value <0.0001. Error bars represent the SEM from three biological replicates. (B) ARF1 KD cells show enhanced sensitivity in a GCA dose-response curve. A GCA or DMSO working solution (200 µM) was serially diluted and co-plated with 20,000 cells per well of ARF1 KD and MSMO1 KD cells in a 96-well plate, with final GCA or DMSO concentrations ranging from 0 – 100 µM. After 48 h, cell viability was measured with CellTiterBlue and the normalized fluorescence, relative to the DMSO-treated samples, was calculated using the smallest and largest mean values to define 0% and 100%, respectively. A dose reponse curve of the normalized fluorescence was plotted against the log10 GCA concentration and IC50 values were calculated using the a four parameter logistic regression model in Graphpad. Error bars represent the SEM from three biological replicates.