Transit helamonster

HeLa cells were transfected with 2.5 μg of DNA using TransIT-HeLa Monster (Mirus Bio) according to the manufacturer’s instructions. Cells were analysed 48 hours after transfection.

mRNA decay assays and Northern blotting were performed as previously described [48] (link). HeLa Tet-off cells (Clontech) were seeded to 12-well plates and cultured in DMEM/10% fetal bovine serum/1% penicillin and streptomycin (full DMEM). siRNAs were transfected at a final concentration of 20 nM using siLentFect Lipid Reagent (Bio-Rad) following the manufacturers protocol. Then either: 1) 24 hours later, plasmid DNA was transfected in the presence of 50 ng/ml tetracycline using TransIt HeLa-Monster (Mirus), or 2) 48 hours later 20 nM siRNA was transfected with plasmid DNA using Lipofectamine 2000 reagent (Inivitrogen) in the presence of 50 ng/ml tetracycline. Two days after plasmid transfection, transcription of the mRNA reporter was activated for six hours with a PBS wash and replacement of full DMEM without tetracycline. Six hours later, transcription was shut off with addition of 1 µg/ml tetracycline. Cells were harvested in Trizol (Invitrogen) for RNA extraction at subsequent time points as indicated, with time “0” taken 20 minutes after tetracycline addition. For each time course, one well of cells were taken up in 2x SDS load buffer (100 mM Tris-HCl pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) for protein analysis by SDS-PAGE and Western blot.

HeLa cells were obtained from ATCC and maintained in DMEM with 10% FBS. Anti-GST antibody was from Santa Cruz (Santa Cruz, CA), anti-HA, anti-FLAG (M2) and anti-β-actin from Sigma-Aldrich (St Louis, MO), anti-GFP/YFP (Ab290) from Abcam (Cambridge, MA), anti-PKD3 and anti-SSH1L from Bethyl Laboratories (Montgomery, TX), anti-pS744/748-PKD (recognizes pS706/710 for human PKD2 and pS731/735 for human PKD3), anti-LIMK, anti-pT508-LIMK, anti-PAK4, anti-pS474-PAK4, anti-Cofilin and anti-pS3-Cofilin from Cell Signaling Technology (Danvers, MA), anti-PKD2 from Millipore (Billerica, MA). The monoclonal antibodies directed against PKD1 and pS978-SSH1L were described and characterized previously [20] (link). Secondary HRP-linked antibodies were from Millipore. Secondary antibodies for immunofluorescence analysis were goat-anti-mouse Alexa Fluor 488 F(ab’)2 and goat-anti-rabbit Alexa Fluor 488 F(ab’)2 from Invitrogen (Carlsbad, CA). TransIT HeLa-Monster (Mirus, Madison, WI) was used for transient transfection of cells.

RDEB/FB/C7 cells, HeLa, Het1a, and Saos2 cells were grown at 37°C with 5% CO₂ in complete DMEM with 10% fetal bovine serum. Plasmids were transfected in HeLa, with TransIT-HeLaMONSTER (Mirus Bio LLC, Madison, WI) or X-tremeGENE9 (Roche, Indianapolis, IN) according to the manufacture's protocols. siRNAs were transfected in HeLa, RDEB/FB/C7, Het1a and Saos2 with Hiperfect (Qiagen, Venlo, Netherlands) according to the manufacture's protocols. In the case of RDEB/FB/C7, siRNAs were transfected twice, on day 0 and on day 1. For lentiviral infection of SLY1-GFP into RDEB/FB/C7 cells, lentiviral particles were produced by co-tranfecting HEK293 cells with a third generation packaging vector pool and pJLM1-SLY1-GFP using TransIT-293 (Mirus Bio LLC). At 72 hr post transfection the viral supernatant was harvested, filtered, and directly added to RDEB/FB/C7 cells.

HeLa cells were transiently transfected with sgRNA, CAS9 and pDonor vector using TransIT HeLa Monster (Mirus). To create a C18orf8-3xMyc knock-in a single donor vector (pDonor C18orf8-3xMyc Hygro, Supplementary Fig. 5a) was used, whereas for HMGCS1-Clover knock-in a combination of three donor plasmids (pDonor HMGCS1-Clover Puro, Hygro, Blast, Supplementary Fig. 1a) was used to select for multiple-allele integration. Transfected cells were transiently selected for sgRNA expression using puromycin at 24–72 h post-transfection, followed by selection of the knock-in cassette using hygromycin or a combination of puromycin, hygromycin and blasticidin at 7 days post-transfection. Selected cells were transiently transfected with Cre recombinase to remove resistance cassettes and single-cell cloned. Single-cell clones were characterised using flow cytometry and Western blotting.

HeLa and HEK293T cells were maintained in RPMI 1640 medium supplemented with 10% FCS. Cell lines were authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) as described recently (Castro et al., 2013). The SNP profiles matched known profiles or were unique. Cells were tested negative for mycoplasma contamination using MycoAlert (Lonza, Switzerland). For transient plasmid transfections, HEK293T cells were transfected with TransIT-293 (Mirus Bio). HeLa cells were transfected with TransIT-HeLaMONSTER (Mirus Bio).
Flp-In T-REx-HeLa cells (generated by Elena Dobrikova and Matthias Gromeier, Duke University Medical Center, Durham, NC, USA) were grown in DMEM containing 10% FCS, 100 µg/mL zeocin, and 10 µg/mL blasticidin (HeLa). These cells stably express the Tet repressor, contain a single Flp Recombination Target (FRT) site, and were used to generate the Flp-In-T-REx-PKD1-GFP lines. Cells were cotransfected with pcDNA5/FRT/TO-PKD1-GFP and the Flp recombinase expression plasmid pOG44 at a ratio of 1:10 and then selected with 500 µg/mL hygromycin B. Induction of protein expression with doxycycline was at 10 ng/mL. Details of the immunofluorescence staining and confocal microscopy can be found in SI Appendix.