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. 2022 Jun 7;11:e74136. doi: 10.7554/eLife.74136

Figure 5. ClC-7 knockout (KO) cells display both lysosomal hyperacidification and swelling reduction.

(A) Agarose gel electrophoresis of PCR product amplified from ClCN7 deletion site of wildtype (ClC7 Wt, left) and CLCN7 KO (ClC-7 KO, right) U2OS cells. (B) EXON1 sequence from CLCN7 WT (black) and KO (purple and teal) alleles. (C) Protocol timeline. Cells were ‘lysosome-loaded’ with Oregon Green 488-dextran (OG) and treated for 3 hr with apilimod (100 nM, red) or its vehicle (0.25% DMSO, control) before imaging. (D) Images from a representative experiment: ClC-7 WT (left) or ClC-7 KO (right) cells acquired by 445 nm laser excitation. Bright objects represent OG-positive lysosomes in control (top) versus apilimod (bottom) conditions. Dotted lines delineate cell outlines. (E, F) Lysosomal pH (E) or size (F) from ClC-7 WT (gray circle) or ClC-7 KO (red triangle) cells in apilimod (filled symbols) versus control (empty symbols) in a representative experiment. Dark symbols are averages over all cells; each pale symbol represents the average lysosomal pH from one cell. p-Values for apilimod effects are obtained from two-way ANOVA. (G) There is no significant difference in pH between untreated WT and untreated KO cells (p=0.5675, unpaired t-test). For (G - I), dark symbols are averages over all experiments; each pale symbol represents the averaged lysosomal pH or size from one experiment (10 and 6 independent experiments for WT and KO conditions, respectively; each experiment represents 8–18 cells per condition). (H, I) Comparison of lysosomal pH shift (H) or size shift (I) induced by apilimod treatment in ClC-7 Wt (gray) versus ClC-7 KO (red) cells. Proton concentration change (H), [H+]change was calculated from apilimod-induced pH shift (∆pH) using the following relation: [H+]change=10−ΔpH. p-Values from unpaired t-test.

Figure 5—source data 1. Raw gel: agarose gel of PCR product amplified from CLCN7 deletion site of wildtype (WT) and CLCN7 knockout (KO) U2OS cells.
Figure 5—source data 2. Labeled gel: same gel as above with labels.

Figure 5.

Figure 5—figure supplement 1. Validation of the U2OS ClC-7 knockout (KO) clone.

Figure 5—figure supplement 1.

(A) RT-qPCR amplification plot obtained from ClC-7 wildtype (Wt) (gray) and ClC-7 KO (red) U2OS cells whole RNA. GAPDH housekeeping RNA level (left gray and left red traces) is used as a control of RNA quantity. ClCN7 RNA level corresponds to right gray and right red traces. ∆ = 3.5 cycles, indicating a 90% decrease in KO ClC-7 RNA level compared to Wt. (B) Agarose gel electrophoresis of qPCR product from ClC-7 Wt (gray) and ClC-7 KO (red) U2OS cells GAPDH or CLCN7 cDNA.
Figure 5—figure supplement 1—source data 1. Raw gel: agarose gel of RT-PCR products of GAPDH (positive control), ClC-7 wildtype (WT), and ClC-7 knockout (KO) U2OS cells.
Figure 5—figure supplement 1—source data 2. Labeled gel: labeled gel of above.
Figure 5—figure supplement 2. .CLCN7 deletion does not affect ‘lysosome-loading’ with Oregon Green 488-dextran (OG) and does not alter pH measurements.

Figure 5—figure supplement 2.

Left: pH calibration curves of U2OS ClC-7 wildtype (WT) or ClC-7 knockout (KO) lysosomes loaded with OG in untreated (control) or 3 hr apilimod-treated conditions (apilimod). Each dot represents the averaged lysosomal 488/445 ratio of one cell (four cells per condition). Right: comparison of cumulative distribution of U2OS ClC-7 Wt (gray, control, 9 cells, 427 objects) versus ClC-7 KO (blue, 10 cells, 548 objects) lysosomal pH population. Each symbol represents the proportion of lysosomes having a pH value below the pH value represented in the abscissa axis.