Figure 5.
Increased RAB7 GTP/GDP ratio in SPG21 KO HeLa cells impairs TFEB phosphorylation by mTOR. (A) Colocalization analysis between endogenously expressed RAB7 and transfected HA-RagC WT in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. The graphs show Mean Manders’ coefficients ± SD. n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment (light gray). The averaged values are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Two-tailed unpaired t test. ***P < 0.001. (B) Western blotting detection of endogenously expressed TFEB in CTRL and SPG21 KO cells transfected for 48 h with siRNAs targeting RAB7. Non-targeting siRNAs (siNT) were used as controls. One representative set of n = 4 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). (C) Western blotting analysis of pTFEB/TFEB ratio 48 h after transfection of SPG21 KO HeLa cells (right panel) or control cells (left panel) with GFP-TFEB and either WT, DN, or CA mCherry-RAB7. α-Tubulin and RAB7 chimeric proteins were detected as controls. Right panel: n = 6 (three independent experiments, including two CTRL clones); left panel: n = 10 for the KO cells (five independent experiments, including two different KO clones) and n = 20 for the CTRL cells (each of the 10 blots contained the two CTRL clones co-transfected with GFP-TFEB and an empty plasmid. One of them is shown in the figure). Mean fold change ± SD. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; ***P < 0.001; ****P < 0.0001. (D) Investigation of GFP-TFEB localization by confocal microscopy in CTRL and SPG21 KO HeLa cells after transfection with the same constructs. Scale bar = 10 µm. The graphs show the quantification of the percentage of cells with predominant nuclear localization for TFEB (upper graph) or the TFEB nuclear/cytoplasmic signal ratio (lower graph). Mean ± SD. For the control conditions transfected with RAB7 constructs, n = 4 (two independent experiments including two different CTRL clones). For the KO conditions, n = 6 (three independent experiments, including two different KO clones). 10 cells were analyzed per clone, as described in A. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.01; ****P < 0.0001. Source data are available for this figure: SourceData F5.

Increased RAB7 GTP/GDP ratio in SPG21 KO HeLa cells impairs TFEB phosphorylation by mTOR. (A) Colocalization analysis between endogenously expressed RAB7 and transfected HA-RagC WT in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. The graphs show Mean Manders’ coefficients ± SD. n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment (light gray). The averaged values are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Two-tailed unpaired t test. ***P < 0.001. (B) Western blotting detection of endogenously expressed TFEB in CTRL and SPG21 KO cells transfected for 48 h with siRNAs targeting RAB7. Non-targeting siRNAs (siNT) were used as controls. One representative set of n = 4 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). (C) Western blotting analysis of pTFEB/TFEB ratio 48 h after transfection of SPG21 KO HeLa cells (right panel) or control cells (left panel) with GFP-TFEB and either WT, DN, or CA mCherry-RAB7. α-Tubulin and RAB7 chimeric proteins were detected as controls. Right panel: n = 6 (three independent experiments, including two CTRL clones); left panel: n = 10 for the KO cells (five independent experiments, including two different KO clones) and n = 20 for the CTRL cells (each of the 10 blots contained the two CTRL clones co-transfected with GFP-TFEB and an empty plasmid. One of them is shown in the figure). Mean fold change ± SD. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; ***P < 0.001; ****P < 0.0001. (D) Investigation of GFP-TFEB localization by confocal microscopy in CTRL and SPG21 KO HeLa cells after transfection with the same constructs. Scale bar = 10 µm. The graphs show the quantification of the percentage of cells with predominant nuclear localization for TFEB (upper graph) or the TFEB nuclear/cytoplasmic signal ratio (lower graph). Mean ± SD. For the control conditions transfected with RAB7 constructs, n = 4 (two independent experiments including two different CTRL clones). For the KO conditions, n = 6 (three independent experiments, including two different KO clones). 10 cells were analyzed per clone, as described in A. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.01; ****P < 0.0001. Source data are available for this figure: SourceData F5.

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