Figure 3.
Activation of RagC by transfection of its GAP FLCN-FNIP2 rescues TFEB phosphorylation and localization. (A) Colocalization analysis between HA-RagC (green) and LAMP2 (red) 24 h after transfection in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. n = 6 (three independent experiments, including two clones per genotype). 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 Manders’ coefficients ± SD. Two-tailed unpaired t test. ns, nonsignificant. (B) Colocalization analysis between HA-RagA and LAMP2 in CTRL and SPG21 KO HeLa cells as described in A. (C) Western blotting analysis of pTFEB/TFEB ratio in CTRL and SPG21 KO HeLa cells 48 h after transfection of GFP-TFEB with either an empty plasmid, a folliculin-His and/or a Flag-FNIP2 construct. α-Tubulin was used as a loading control. Folliculin and FNIP expressions were assessed using anti-His and anti-Flag antibodies, respectively. The graph shows the quantifications of the mean fold change in pTFEB/TFEB ratio ± SD in three independent experiments for two KO clones (n = 6). n = 12 for the CTRL cells (each of the six blots contained the two CTRL clones co-transfected with GFP-TFEB and an empty plasmid. One of them is shown in the figure). Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01. (D) Confocal microscopy detection of GFP-TFEB distribution in CTRL and SPG21 KO HeLa cells 72 h after transfection with folliculin-His (red) and/or Flag-FNIP2 (blue) constructs. Scale bar = 10 µm. The graphs show the percentage of cells with predominant nuclear localization of TFEB (left) or the TFEB nuclear/cytoplasmic signal ratio (right). Mean ± SD. n = 6 (three independent experiments for two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment, as described in (B). Two-tailed unpaired t test. ns, non-significant; ****P < 0.0001. (E) Western blotting detection of endogenously expressed TFEB in CTRL and SPG21 KO cells transfected with a Flag-FNIP2 construct for 48 h. One representative set of four independent experiments is shown. The black arrowhead indicates the higher MW forms of TFEB (pTFEB). The white arrowhead indicates lower MW forms of TFEB (non-phosphorylated). Source data are available for this figure: SourceData F3.

Activation of RagC by transfection of its GAP FLCN-FNIP2 rescues TFEB phosphorylation and localization. (A) Colocalization analysis between HA-RagC (green) and LAMP2 (red) 24 h after transfection in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. n = 6 (three independent experiments, including two clones per genotype). 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 Manders’ coefficients ± SD. Two-tailed unpaired t test. ns, nonsignificant. (B) Colocalization analysis between HA-RagA and LAMP2 in CTRL and SPG21 KO HeLa cells as described in A. (C) Western blotting analysis of pTFEB/TFEB ratio in CTRL and SPG21 KO HeLa cells 48 h after transfection of GFP-TFEB with either an empty plasmid, a folliculin-His and/or a Flag-FNIP2 construct. α-Tubulin was used as a loading control. Folliculin and FNIP expressions were assessed using anti-His and anti-Flag antibodies, respectively. The graph shows the quantifications of the mean fold change in pTFEB/TFEB ratio ± SD in three independent experiments for two KO clones (n = 6). n = 12 for the CTRL cells (each of the six blots contained the two CTRL clones co-transfected with GFP-TFEB and an empty plasmid. One of them is shown in the figure). Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01. (D) Confocal microscopy detection of GFP-TFEB distribution in CTRL and SPG21 KO HeLa cells 72 h after transfection with folliculin-His (red) and/or Flag-FNIP2 (blue) constructs. Scale bar = 10 µm. The graphs show the percentage of cells with predominant nuclear localization of TFEB (left) or the TFEB nuclear/cytoplasmic signal ratio (right). Mean ± SD. n = 6 (three independent experiments for two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment, as described in (B). Two-tailed unpaired t test. ns, non-significant; ****P < 0.0001. (E) Western blotting detection of endogenously expressed TFEB in CTRL and SPG21 KO cells transfected with a Flag-FNIP2 construct for 48 h. One representative set of four independent experiments is shown. The black arrowhead indicates the higher MW forms of TFEB (pTFEB). The white arrowhead indicates lower MW forms of TFEB (non-phosphorylated). Source data are available for this figure: SourceData F3.

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