Fig. 4: CTSD deficiency expands the acidic cell compartment of short-term starved tumor cells. | Nature Communications

Fig. 4: CTSD deficiency expands the acidic cell compartment of short-term starved tumor cells.

From: Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

Fig. 4

a Flow cytometry analysis of LysoTrackerTM Green intensity in Ctsd+/+ and Ctsd−/− PyMT cells cultured for 7–10 days in 10% FCS or 1% FCS medium or treated with Torin (n = 3 independent experiments for Torin, n = 6 independent experiments for the rest; two-sided three-way ANOVA for the factors intensity of LysoTrackerTM, Ctsd genotype and FCS). b Analysis of LAMP1 expression by Western blot (with TUBA as loading control) in 10% FCS and 1% FCS Ctsd+/+ and Ctsd−/− PyMT cells. Slight molecular weight changes due to different glycosylation pattern. Representative of 2 independent experiments. Quantification is below (n = 1 experiment). c Relative Lamp1 expression determined by RT-PCR in 10% FCS and 1% FCS Ctsd+/+ and Ctsd−/− PyMT cells (n = 3 independent experiments; two-sided two-sample t-test). Activity assay with substrates specific for aspartic (d) or cysteine (e) proteases in 10% FCS and 1% FCS Ctsd+/+ and Ctsd−/− PyMT cells (n = 2 independent experiments). f Lysosomal activity assay using a quenched fluorogenic substrate in 10% FCS and 1% FCS Ctsd+/+ and Ctsd−/− PyMT cells (n = 4 independent experiments for 10% FCS, n = 3 independent experiments for 1% FCS). Histogram including controls blocking the endocytic substrate uptake (Cytochalasin D) or lysosomal acidification (Bafilomycin A1) on the left, median fluorescence intensity (MFI) on the right. g Analysis of CTSL expression by Western blot (with TUBA as loading control) in 10% FCS and 1% FCS Ctsd+/+ and Ctsd-/- PyMT cells. Pro, zymogen; SC, single-chain form; HC, heavy chain. Representative of 2 independent experiments. Quantification is to the right (n = 2 independent experiments). h Relative Ctsl expression determined by RT-PCR in 10% FCS and 1% FCS Ctsd+/+ and Ctsd−/− PyMT cells (n = 3 independent experiments; two-sided two-sample t-test). i Analysis of CTSB expression by Western blot (with TUBA as loading control) in 10% FCS and 1% FCS Ctsd+/+ and Ctsd−/− PyMT cells. Pro, zymogen; SC, single-chain form. Representative of 2 independent experiments. Bar charts show all data points with mean + SD and p-value. Source data are provided as a Source Data file.

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