Supplementary Figure 6: Depletion of UBXN10 inhibits cilia formation.
(a) LLC-PK1 GFP-UBXN10 cells were transfected with siRNAs targeting VCP or UBXN10. Cells were induced to ciliate by serum deprivation, fixed and stained with acetylated tubulin. The number of ciliated cells and the number of GFP-UBXN10 positive cilia were quantified. n = 131 (Control), 124 (siUBXN10-3), 156 (siVCP-7) cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance was calculated using unpaired, two-tailed Students t-test. P values compared to Control are shown. ∗/∗∗, P ≤ 0.05, 0.01 respectively. (b) UBXN10 was stably depleted in hTERT-RPE1 using two separate pLKO-based short hairpin RNAs. Cells were selected with Puromycin, induced to ciliate and the number of ciliated cells was quantified based on acetylated tubulin staining. n = 98 (shGFPl), 115 (sh114), 103 (sh909) cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance was calculated using unpaired, two-tailed Students t-test. P values compared to shGFP are shown. ∗/∗∗, P ≤ 0.05, 0.01 respectively. (c) UBXN10 knockout hTERT-RPE1 cells generated via CRISPR-CAS9 have impaired ciliogenesis. 40X images showing a larger field of view. Expression of GFP-UBXN10 in the knockout cell line reinstates ciliogenesis. Cells were stained with pericentrin and acetylated. Scale bar is 10 μm. (d) Cells were treated with either 50 μM NMS-873 for 5 min or 5 μM NMS-873 for 4 h, extracts were generated and subjected to SDS-PAGE and immunoblotting with α-ubiquitin antibodies.
