|
In vitro assays indicate that CEP76 variants confer the loss of function. (A) Representative high-resolution confocal images of nuclei along with centrosomes are shown for each transfected condition. U20S cells were cotransfected with centrosomal markers CETN1 [GFP (green fluorescent protein)–tagged] and CEP76 (mCherry-tagged), followed by independent siRNA transfections (siNS or siCEP76, a siRNA targeting 3′UTR of endogenous CEP76-NM_024899.4). An empty mCherry-vector backbone was used as a vehicle control. Nuclei are labeled in blue with Hoechst 34580. For each condition, the representative merged image is shown on the left, and zoomed images of centrioles/basal bodies, as indicated in dotted boxes, are shown on the right. A common variant in CEP76 (p.Ser172Asn) was used as a population control. Scale bar, 10 μm. All images are scaled to the same magnification. (B) Bar graphs show the percentage of cells with more than four centrin dots in transfected U20S cells (n = 140 to 220 transfected cells per condition; replicated twice). Statistical significance was calculated using Fisher’s exact test, and P values are shown (P vs siCEP76 represents the P value against CEP76 knockdown, and P vs WT represents P values against WT CEP76 rescue). Error bars represent 95% confidence interval. ORF, open reading frame; ns, not significant; (−), not present; (+), present.
|
