(A) Top panels: immunoblots show Piwil1 knockdown efficiency. Bottom panels: cell viability of 4121 and 3832 GSCs after Piwil1 knockdown (n = 4 replicates for each group).
(B) Top panels: representative images of tumorspheres in control shNT and shPiwil1 #1 and #2 GSCs. Scale bar, 200 μm. Bottom panels: quantification of tumor sphere numbers after Piwil1 knockdown (n = 12 replicates each group).
(C) Left panels: immunofluorescence imaging of EdU incorporation in 4121 and 3832 GSC tumorspheres after Piwil1 knockdown. Right panel: quantification of the fraction of EdU+ cells (n > 1,000 cells for each group).
(D) Western blot of cleaved PARP and cleaved caspase 3 proteins in GSCs after Piwil1 knockdown.
(E and F) in vitro limiting dilution assays of 4121 (E) and 3832 (F) GSCs after Piwil1 knockdown. Tables on the right show the estimated stem cell frequencies in control shNT and shPiwil1 knockdown groups.
***p < 0.001; ****p < 0.0001. Data are represented as mean ± SD. See also Figures S3 and S4.