Supplementary Figure 7: Inhibition of VCP leads to destabilization of IFT-B and altered rates of trafficking within the primary cilium.
(a) Acute treatment with 50 μM NMS-862 or NMS-873 leads to an increase in both anterograde and retrograde IFT-88 EYFP velocities. Treatment with 5 μM NMS-862 or NMS-873 for short (<5 min) time periods does not produce the same phenotype. The fluorescence image time series was subjected to kymograph analysis in ImageJ. n (anterograde, retrograde) from left to right = 54, 56, 61, 63, 50, 62, 53, 60, 68, 56 cells pooled from 2 independent experiments. Error bars represent mean ± s.e.m. Statistical significance calculated using ANOVA, P values compared to DMSO for anterograde or retrograde transport are shown. N.S., not significant. ∗/∗∗, P ≤ 0.05, 0.01 respectively. (b) Treatment of LLC-PK1 cells with NMS-873 leads to a shortening of cilia over time. Cells were treated with 5 μM NMS-873 for indicated times and stained for acetylated tubulin. The length of the cilia were measured based on acetylated tubulin staining on MetaMorph. (c) Kymographs derived from TIRF microscopy of IMCD EYFP-IFT88/UBXN10-mCHERRY treated with DMSO or 50 μM NMS-873 for <5 min. Only cilia that displayed motile EYFP-IFT88 particles were measured for this study. The fraction of these cilia that also exhibited observable UBXN10-Cherry motility was then determined. Treatment with NMS-873 resulted in a loss of UBXN10 trafficking within cilia. n = 75 (DMSO) and 82 (NMS-873). Scale bars in time (2 s) and distance (2 μm) are shown. (d) Inhibition of VCP leads to destabilization of the IFT-B complex. Ciliated hTERT RPE1 cells were treated with 5 μM of NMS-873 for 4 h. Cell lysates were fractionated by gel filtration, resolved by SDS PAGE and probed for the indicated proteins. Treatment with NMS-873 causes CLUAP1 destabilization from higher molecular weight fractions. (e) hTERT-RPE1 cells were transfected with siRNAs to IFT25 or IFT27. Lysates were resolved on SDS PAGE and immunoblotted to show antibody specificity and observed molecular weights of the IFT-25 and IFT27. (f) LLC-PK1 cells were treated with 5 μM of NMS-873 in a time course experiment. Cell lysates were resolved on SDS PAGE and immunoblotted with the indicated antibodies. UBXD adaptor levels begin to decline at 4 h post treatment. (g) GFP-UBXN10 LLC-PK1 cells were treated with 5 μM of NMS-873 in a time course experiment. Cell were fixed and stained for GFP and acetylated tubulin and the intensity of GFP-UBXN10 within cilia was determined using Metamorph. Treatment with NMS-873 causes a decrease in GFP-UBXN10 in cilia within an hour. n = 56 (DMSO), 72 (1 h), 45 (2 h), 31 (4 h) cells pooled from 2 independent experiments. Maxima, centre, minima and quartiles (Q1,2,3) for each sample from left to right: (68, 33, 8, 14, 20, 29), (38, 16, 6, 9, 12, 19) (29, 13, 3, 7, 12, 17), (21, 9, 3, 8, 9, 18). Error bars represent mean ± s.d. Statistical significance calculated using ANOVA, P values compared to DMSO are shown. ∗∗P ≤ 0.01.
