Figure S4.

Loss of clh-6 affects endocytic cargo degradation. (A–B′′) DIC, confocal fluoresence, and merged images of wild-type (A, A', and A'') and clh-6(tm617) (B, B', and B'') adults expressing CAV-1::GFP. White arrowheads indicate the spermatheca; yellow arrowheads indicate fertilized and dividing embryos. The percentage of worms with the representative pattern is quantified and shown at the lower right corner in each panel. 13 animals (n) were scored in each strain. (C–D′′) DIC and confocal fluorescence images of a coelomocyte in wild-type adults expressing GFP secreted from body wall muscle cells (Secretory GFP). Worms were treated with DMSO (C–C′′) or Pitstop 2 (D–D′′). GFP is endocytosed into the coelomocyte (outlined by the dashed line) in DMSO- but not Pitstop 2–treated worms. (E) The purity of lysosomes obtained by LysoIP was examined by Western blot using antibodies that detect the lysosomal reporter SCAV-3::RFP::3xHA (anti-HA) and recognize proteins in lysosomes (anti-SCAV-3, anti-ASP-4), mitochondria (anti-HSP-60), ER (anti-PDI), endosomes (anti-RAB-5) or nuclei (anti-LAMIN). 1% of the whole worm lysate and 2% of the purified lysosomal prep were loaded. EE, early endosomes. (F–F′′) DIC and confocal fluorescence images of the hypodermis in wild type expressing the lysosomal markers SCAV-3::RFP::3xHA and SCAV-3::GFP. The RFP signal overlaps well with SCAV-3::GFP (white arrowheads). (G–G′′) The integrity of lysosomes purified by LysoIP was examined by LysoSensor green staining. SCAV-3::RFP::3xHA-positive lysosomes are well stained by LysoSensor green (white arrowheads). Scale bars in A–D′′ and F–G′′ represent 5 µm. (H–J) The potassium concentration in lysosomes purified from multi-tissues (H) or the hypodermis of C. elegans (I and J) was determined as described in the Materials and methods. Relative potassium levels are presented. At least four independent experiments were performed, and data are shown as mean ± SD. Unpaired two-tailed Student’s t test was performed to compare mutant datasets with wild type. All points had P > 0.5. Source data are available for this figure: SourceData FS4.