Figure 2.
The decreased pTFEB/TFEB ratio in SPG21 KO HeLa cells is due to defective TFEB presentation to mTORC1 by RagC. (A) Colocalization analysis between mTOR (green) and LAMP1 (red) in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. n = 6 (three independent experiments each, including two different CTRL and two different 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. Data are represented as mean Manders’ coefficients ± SD. Two-tailed unpaired t test. ns, nonsignificant. (B) Western blotting detection of the phosphorylated form (on threonine 389) and total form of p70S6K in CTRL and SPG21 KO HeLa cells. α-Tubulin is shown as a loading control. The graph shows the quantification of the mean fold change ratio of p-p70S6K/p70S6K ± SD in 7 independent experiments for both CTRL and both KO clones (n = 14). Two-tailed unpaired t test. ns, nonsignificant. (C) Western blotting analysis of pTFEB/TFEB ratio in CTRL and SPG21 KO cells 24 h after transfection of GFP-TFEB with WT or CA forms of HA-RagA or HA-RagC constructs. Rag proteins were detected with an anti-HA antibody, and α-tubulin is shown as a loading control. n = 8 for the KO cells (four independent experiments, including two different KO clones and n = 16 for the CTRL cells [each of the eight blots contained the two CTRL co-transfected with GFP-TFEB and an empty plasmid. One of them is shown in the figure]). Mean fold change of pTFEB/TFEB ratio ± SD. Two-tailed unpaired t test. ns, nonsignificant; **P < 0.01; ***P < 0.001. (D) Confocal microscopy detection of GFP-TFEB in CTRL and KO clones after co-transfection with WT or CA HA-RagA or HA-RagC constructs (red). Scale bar = 10 µm. The graphs show the quantification of the percentage of cells with predominant nuclear signal for TFEB (top graph) or the TFEB nuclear/cytoplasmic signal ratio (bottom graph). Three independent experiments that included two CTRL and two KO clones (n = 6). 10 cells were analyzed per clone in each biological replicate. Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant; ****P < 0.0001. (E) Western blotting detection of endogenously expressed TFEB in CTRL and SPG21 KO cells (two clones each) transfected for 48 h with WT or CA HA-RagA or C. 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 F2.

The decreased pTFEB/TFEB ratio in SPG21 KO HeLa cells is due to defective TFEB presentation to mTORC1 by RagC. (A) Colocalization analysis between mTOR (green) and LAMP1 (red) in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. n = 6 (three independent experiments each, including two different CTRL and two different 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. Data are represented as mean Manders’ coefficients ± SD. Two-tailed unpaired t test. ns, nonsignificant. (B) Western blotting detection of the phosphorylated form (on threonine 389) and total form of p70S6K in CTRL and SPG21 KO HeLa cells. α-Tubulin is shown as a loading control. The graph shows the quantification of the mean fold change ratio of p-p70S6K/p70S6K ± SD in 7 independent experiments for both CTRL and both KO clones (n = 14). Two-tailed unpaired t test. ns, nonsignificant. (C) Western blotting analysis of pTFEB/TFEB ratio in CTRL and SPG21 KO cells 24 h after transfection of GFP-TFEB with WT or CA forms of HA-RagA or HA-RagC constructs. Rag proteins were detected with an anti-HA antibody, and α-tubulin is shown as a loading control. n = 8 for the KO cells (four independent experiments, including two different KO clones and n = 16 for the CTRL cells [each of the eight blots contained the two CTRL co-transfected with GFP-TFEB and an empty plasmid. One of them is shown in the figure]). Mean fold change of pTFEB/TFEB ratio ± SD. Two-tailed unpaired t test. ns, nonsignificant; **P < 0.01; ***P < 0.001. (D) Confocal microscopy detection of GFP-TFEB in CTRL and KO clones after co-transfection with WT or CA HA-RagA or HA-RagC constructs (red). Scale bar = 10 µm. The graphs show the quantification of the percentage of cells with predominant nuclear signal for TFEB (top graph) or the TFEB nuclear/cytoplasmic signal ratio (bottom graph). Three independent experiments that included two CTRL and two KO clones (n = 6). 10 cells were analyzed per clone in each biological replicate. Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant; ****P < 0.0001. (E) Western blotting detection of endogenously expressed TFEB in CTRL and SPG21 KO cells (two clones each) transfected for 48 h with WT or CA HA-RagA or C. 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 F2.

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