Supplementary Figure 2: CRISPR–cas9 SWELL1 gene ablation. Related to Fig. 2.

(a) Sequences of mutant alleles showing deletions generated by CRISPR–cas9 single-guide RNA (gRNA) mediated approach (KO1). (b) CRISPR–cas9 double-gRNA mediated SWELL1 knockout (KO2) resulting in excision of the DNA fragment (240 bp) between the two cut sites. (c) PCR of the double-gRNA mediated knockout gene yields a ∼639 bp amplicon, reflecting the expected 240 bp deletion. (d) Schematic representation of CRISPR–cas9 mediated loxP knockin around Exon 3 to generate SWELL1^fl allele. SWELL1^null allele is generated by Cre-Lox mediated excision of Exon 3. (e) Cre-mediated Exon 3 deletion yields the expected PCR amplicons 426 and 196 bp from two individual primer pairs flanking loxP sites around Exon 3. The ∼5 Kb region between loxP sites in the SWELL1^fl allele could not be amplified. Uncropped blots for c are shown in Supplementary Fig. 9a–d, and e in Supplementary Fig. 9a–e. Representative gel image from 2 independent experiments (c,e). (f) Experimental approach for generating WT and SWELL1 KO primary SVF used for differentiation into cultured primary adipocytes. (g) Bright-field image (left) of SWELL1^fl primary cultured adipocytes from SVF and mCherry fluorescence image (right) showing nuclear localization of Cre-mCherry. Scale bar: 200 μm. (h) Relative mRNA expression assessed by qPCR (n = 3 each). (i–k) Representative VRAC current over time ± hypotonic swelling (i), VRAC current-voltage plots upon swelling (j), and mean peak outward (+100 mV) and inward (−100 mV) VRAC current density (k) in WT and SWELL1 primary SVF (n = 5–6 each). Significance between the indicated groups in h and k were calculated using a two-tailed Student’s t-test. Exact P-values are listed in Supplementary Table 6. Error bars represent mean ± s.e.m.^∗(P < 0.05), ^∗∗(P < 0.01).