Dharmafect 2 transfection reagent

Prior to transfection, cells were starved for 6 h in order to achieve proper cell cycle synchronization. T98G cancer cells were transfected overnight with Dharmacon's chemically synthesized siRNA SMARTpools [human PC‐1, L‐007666‐00‐0005, ON‐TARGETplus Human PKD1 (5310) siRNA–SMARTpool, 5 nmol] and non‐targeting siRNA for control cells (D‐001210‐01‐05, siGENOME Non‐Targeting siRNA #1, 5 nmol), in dilution 1:20 in 1× siRNA buffer, using DharmaFECT 2 Transfection Reagent, 0.2 ml (Dharmacon) in dilution 1:50 in DMEM (Gibco, Thermo Fisher Scientific).18 (link), 19 (link) After 16 h, the medium was changed and the cells were treated with IgPC1 and/or HP and cultured for 24 and 48 h.

After a 24-hour incubation with antibiotic-free medium, cells were transfected with anti-GIPC small interfering RNA (siRNA) using the DharmaFECT 2 Transfection Reagent (Dharmacon, Lafayette, CO). Seventy-two to 96 h after transfection, GIPC knockdown was confirmed by Western blot analysis. A similar siRNA approach was adopted for anti-Atg7 and anti-Beclin1 knockdown. For glucose starvation experiments, both control siRNA and GIPC siRNA treated AsPC-1 cells were kept in glucose free RPMI supplemented with 10% FBS for final 16 h of the 96 h experiment. For autophagic flux experiments, both control siRNA and GIPC siRNA treated AsPC-1 and PANC-1 cells were treated with indicated concentrations of Pepstatin-A and E-64d for final 24 h of the 96 h experiment.

The screening of the LNA library (Exiqon, Woburn, MA, USA) was performed as previously described [16 (link)]. Briefly, SW480 cells (2 × 10³ cells/well) were seeded in 96-well plates. DharmaFECT 2 Transfection Reagent (Dharmacon, Lafayette, CO, USA) was diluted in Optimem medium (Thermo Fisher Scientific, Waltham, MA, USA), seeded on LNAs and incubated for 15–20 min at room temperature. The transfection mixture was then added to the cells at the final LNA concentration of 25 nM. These conditions were formerly set up to ensure ~90% transfection efficiency. The percentage of transfected cells was monitored with a control siRNA against NF1b mRNA (40 nM), and verified by Real-Time PCR. As a positive control, the siRNA against PLK-1 was added on empty wells of the library plates. Cancer cells were assayed 72 h post-transfection with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) on a Beckman DTX 880 plate luminometer (Beckman Coulter, Brea, CA, USA). The relative viability was calculated and normalized to the average of all samples, excluding positive controls. The experiment was performed in triplicate. Values below and above twofold the standard deviation were considered significant and were used in further analyses.

A siGENOME library (Dharmacon, Lafayette, CO, USA) containing a pool of 4 distinct siRNA duplexes (6.25 pmol) in each well that target a specific candidate gene or in separate wells, positive and negative controls [6 (link),7 (link)], was employed in the scQuantIM screen. Standard reverse transfection with DharmaFECT 2 transfection reagent (Dharmacon, Lafayette, CO, USA) was performed according to manufacturer’s instructions. Cells were seeded, allowed to grow for 4 (HCT116) to 6 days (hTERT) at 37 °C, fixed (4% paraformaldehyde) and counter-stained (Hoechst 33342) to visualize nuclei [7 (link)]. Subsequent direct tests employed ON-TARGETplus siRNA duplexes (Dharmacon, Lafayette, CO, USA) in a pooled format as detailed elsewhere [6 (link)]. Gene silencing was confirmed by Western blot 4 days post-transfection as described [23 (link)]. Membranes were blotted with the primary antibody at the indicated dilutions (Table 1) and visualized using secondary antibodies conjugated to horseradish peroxidase. Blots were imaged on a MyECL Imager (Thermo Scientific, Mississauga, ON, Canada) using standard chemiluminescence.