GoKawiila. Representative confocal images of HA-CTSA and LAMP1 in AML12 cells. Cells were transfected with HA-CTSA prior to staining. Co-localization was assessed using ImageJ, and Pearson’s correlation coefficient was calculated as r = 0.818. Scale bar, 10μm. b. Representative immunoblots of GTPγS/GDP loading assay showing the interaction between HA-CTSA and GTPγS- or GDP-loaded FLAG-RalA. Purified FLAG-RalA was preloaded with either GTPγS or GDP to mimic the active or inactive state of RalA, respectively, and used as bait to pull down HA-CTSA from HEK293T cell lysates. IB, immunoblot. c. Representative confocal images of RalA, CTSA, and HA-Tmem192 in AML12 cells. Cells were infected with lentiviruses expressing HA-Tmem192. Scale bar, 2μm. d, e. Representative immunoblots (d) and quantification of the CTSA-lyso/CTSA-pre ratio (e) in liver tissues from C57BL/6J mice after 4 weeks on NCD or HCD. CTSA-pre, CTSA precursor; CTSA-lyso, lysosomal CTSA. f. Representative confocal images of CTSA and HA-Tmem192 in WT or RalGAPB-KO AML12 cells. Cells were infected with lentiviruses expressing HA-Tmem192. Pearson’s correlation coefficients are indicated in each image. Scale bar, 5μm. g. Representative immunoblots of liver tissues from the indicated mice after 20 weeks on HCD. n = 4 mice per group. h. Plasma CTSA levels from the indicated mice after 20 weeks on HCD. n = 12 mice per group. i. Representative immunoblots of CTSA in culture supernatants and cell lysates from WT and RalGAPB-KO AML12 cells. The relative amount of CTSA in supernatants (sup) normalized to CTSA in corresponding lysates is shown as the ratio below each blot. n = 2 per group. j. CTSA levels in supernatants from WT and RalGAPB-KO AML12 cells. n = 3 per group. k. Representative immunoblots in AML12 cells. Cells were infected with lentiviruses expressing pLEX-HA or HA-CTSA prior to harvest. n = 3. l. Representative immunoblots in WT and RalGAPB-KO (KO) AML12 cells treated with DMSO or 100 μM AEBSF overnight in LPDS medium. n = 2. m, n. Representative confocal images (m) and quantification of plasma membrane LDLR fluorescence intensity (n) in the indicated non-permeabilized AML12 cells. Scale bar, 10 μm. n = 32/27/47/37 cells. o, p. Representative confocal images (o) and quantification of DiI-LDL fluorescence intensity (p) in the indicated AML12 cells. Cells were cultured overnight in LPDS medium, then incubated with 10 μg ml−1 DiI-LDL for two hours to assess LDL uptake. Scale bar, 10 μm. n = 100/58/92/91 cells. q. Relative mRNA levels of Ldlr in livers from control (sgLacZ, n = 9) or CTSA deletion (sgCTSA, n = 7) mice. r-u. Body weight (r), liver-to-body weight ratio (%) (s), plasma glucose (t), and plasma TG (u) from control (sgLacZ) or CTSA deletion (sgCTSA) mice. sgLacZ: n = 9/9/9/8; sgCTSA: n = 7. v, w. Quantification of LysoTracker number (v) and fluorescence intensity (w) per cell in control (sgLacZ) or CTSA deletion (sgCTSA) primary hepatocytes. n = 15 cells. Data are mean ± s.e.m. Each dot (n) represents one biological replicate. Unpaired two-sided Student’s t-test (e, h, j, q-w); one-way ANOVA with Tukey’s test (n, p). ns, not significant.
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