Figure 8.
The KO of SPG21 slightly increases the expression of some CLEAR genes and promotes the anterograde transport of lysosomes in HeLa cells. (A) RT-qPCR analysis of the expression of genes belonging to the CLEAR network in CTRL and SPG21 KO cells: CTSB, CTSC, CTSD, GUSB, HEXA, HEXB, LAMP1, and MANBA. LAMP2 was included as a control. The graph shows the relative mRNA expression of these genes normalized to GAPDH expression. n = 6 (three independent experiments with two CTRL [open circles: CTRL1; open squares: CTRL2] and two KO clones [black triangles: KO1; black diamonds: KO2]). Mean fold changes ± SD. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01. (B) Investigation of lysosome number and distribution in CTRL and SPG21 KO HeLa cells. Lysosomes were detected with an anti-LAMP1 antibody (green). Scale bar = 10 µm. Graphs represent the number of lysosomes per cell (left graph) and lysosome distribution (right graph). Lysosome distribution was determined by drawing four circles around the nucleus at 5-µm intervals and by calculating the relative LAMP1 fluorescence intensity in each interval as shown in the schematic representation. n = 6 (three independent experiments with 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 < 0.05. (C) Analysis of lysosome distribution in CTRL and SPG21 KO cells 48 h after co-transfection with an mCherry-RAB7 construct and with an empty plasmid or an SPG21 WT-myc-flag construct (blue) (note that this is the same experiment presented in Figure 4D, but the data is now analyzed for LAMP distribution). Lysosomes were detected with an anti-LAMP1 antibody (green). Scale bar = 10 µm. The graph shows the quantifications of lysosome distribution in cells overexpressing mCherry-RAB7 as explained in B. n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone, as described in B. Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant; **P < 0.01; ***P < 0.001; ****P < 0.0001.

The KO of SPG21 slightly increases the expression of some CLEAR genes and promotes the anterograde transport of lysosomes in HeLa cells. (A) RT-qPCR analysis of the expression of genes belonging to the CLEAR network in CTRL and SPG21 KO cells: CTSB, CTSC, CTSD, GUSB, HEXA, HEXB, LAMP1, and MANBA. LAMP2 was included as a control. The graph shows the relative mRNA expression of these genes normalized to GAPDH expression. n = 6 (three independent experiments with two CTRL [open circles: CTRL1; open squares: CTRL2] and two KO clones [black triangles: KO1; black diamonds: KO2]). Mean fold changes ± SD. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01. (B) Investigation of lysosome number and distribution in CTRL and SPG21 KO HeLa cells. Lysosomes were detected with an anti-LAMP1 antibody (green). Scale bar = 10 µm. Graphs represent the number of lysosomes per cell (left graph) and lysosome distribution (right graph). Lysosome distribution was determined by drawing four circles around the nucleus at 5-µm intervals and by calculating the relative LAMP1 fluorescence intensity in each interval as shown in the schematic representation. n = 6 (three independent experiments with 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 < 0.05. (C) Analysis of lysosome distribution in CTRL and SPG21 KO cells 48 h after co-transfection with an mCherry-RAB7 construct and with an empty plasmid or an SPG21 WT-myc-flag construct (blue) (note that this is the same experiment presented in Figure 4D, but the data is now analyzed for LAMP distribution). Lysosomes were detected with an anti-LAMP1 antibody (green). Scale bar = 10 µm. The graph shows the quantifications of lysosome distribution in cells overexpressing mCherry-RAB7 as explained in B. n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone, as described in B. Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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