Dharmacon on targetplus smartpool sirnas

HRMECs were transfected with Dharmacon ON-TARGETplus SMARTpool siRNAs specifically targeting human CAMK2G (catalog L-004536-00) or CAMK2D (catalog L-004042-00) isoforms for 24 hours using DharmaFECT reagent (Thermo Fisher Scientific). Dharmacon ON-TARGETplus siCONTROL Nontargeting siRNA (catalog D-001810-10) was used as control siRNA. Quantitative PCR (qPCR) was performed to determine knockdown efficiency as previously described (66 (link)). Primers (Integrated DNA Technologies) were designed to amplify human CAMK2G (forward primer 5′-TCCTGTATATCCTCCTGGT-3′, reverse primer 5′-CATCTGGTTGATCAAGTTC-3′) and human CAMK2D (forward primer 5′-GGATCTGTCAACGTTCTACT-3′, reverse primer 5′-TGTGGATTACAGTAGTTTGG-3′), which were quantified relative to GAPDH (human, forward primer 5′-GAGTCAACGGATTTGGTCGT-3′, reverse primer 5′-GACAAGCTTCCCGTTCTCAG-3′). Cells were transfected with 25 nM siRNA, a concentration that was found to consistently produce ≥80% transcript knockdown for each CAMKII isoform and where knockdown of CAMK2G had no effect on CAMK2D mRNA expression and vice versa (Supplemental Figure 6).

For siRNA-mediated RNA interference, cells were transfected with 100 nM Dharmacon-ONTARGETplus SMARTpool siRNAs (CACNA1C L-006123-00, CACNA1H L-006128-00, non-targeting control D-001810-10-05; Thermo Fisher Scientific, Waltham, MA, USA) 24 h after plating as previously described [28 (link)], 24 h after siRNA treatment cells were incubated with SRM overnight followed by stimulation with TGFβ1 as described above.

Silencing of human USP10 and USP13 expression was performed using Dharmacon ON-TARGETplus SMARTpool siRNAs (USP10: L-006032-00-0010; USP13: L-006064-00-0010, Horizon Discovery, Cambridge, United Kingdom). The non-targeting control pool (D-001810-10-50, Horizon Discovery) was used as control. The siRNAs (20 nM) were transfected using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) in accordance with the manufacturer’s reverse transfection protocol. After 24 h or 48 h, the cells were used for further experiments. Because PLK1 knockdown caused severe cell death, PLK1 siRNAs (L-003290-00-0005, Horizon Discovery) was used as an experimental control for transfection.