pTFEB/TEFB ratios in control cells after transfection of RagA, RagC, folliculin/FNIP, or TBC1D5 constructs, and analysis of the localization of endogenous folliculin when Flag-FNIP2 is overexpressed. (A, C and D) Western blotting analysis of pTFEB/TFEB ratio in CTRL HeLa cells 24 h (RagA/RagC) or 48 h (folliculin/FNIP; TBC1D5) after transfection of an empty plasmid or GFP-TFEB with either an empty plasmid, HA-RagA (WT or CA), HA-RagC (WT or CA), FLCN-His, and/or Flag-FNIP or TBC1D5-HA constructs. α-Tubulin was used as a loading control. The graphs show the mean fold change in pTFEB/TFEB ratio ± SD. For RagA and RagC overexpression experiments, n = 6 (three independent experiments with two different CTRL clones (open circles: CTRL1; open squares: CTRL2); for folliculin/FNIP and TBC1D5 overexpression experiments, n = 4 (two independent experiments with two different CTRL clones). Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05. (B) Analysis of endogenous folliculin (green) distribution by confocal microscopy in CTRL and SPG21 KO HeLa 48 h after the overexpression of a Flag-FNIP2 plasmid (red) or an empty plasmid. Scale bar = 10 µm. The graph shows the quantifications of folliculin granularity. n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment. These values are shown in light gray. Their averages are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant. **P < 0.01. Source data are available for this figure: SourceData FS2.