Fig. 1: The elevated CMA activity in GSCs correlates with lysosomal LAMP2A abundance.

a Representative images (left) and quantitative analysis (right) of CMA flux, indicated by the number of KFERQ puncta, in GSC and non-GSC cells stably expressing PAmCherry-KFERQ or PAmCherry-KFSDA (a mutant KFERQ sequence that cannot be recognized by HSC70). n = 40 randomly selected cells. Scale bar, 10 μm. b In vitro binding and uptake assay of GAPDH by the lysosome in GSC and non-GSC cells. n = 3 independent experiments. c Immunoblotting (IB) analyses for indicated proteins after treating with 10 μM MG132, 5 mM 3-MA, or LN (10 μM leupeptin and 20 mM NH₄Cl) in GSC cells (M83 and 23) and non-GSC cells (LN229 and U251), respectively. Band intensities of indicated proteins were quantified using Image J, normalized to β-actin.n = 3 independent experiments. d Representative images (left) and quantification (right) of CMA activity detection by KFERQ puncta numbers in GSCs (M83 and 456) and their corresponding DGCs. n = 40 randomly selected cells. Scale bar, 10 μm. e IB analyses for LAMP2A and CMA-associated chaperone proteins (HSC70, HSP40 and HSP90) in lysosomes isolated from the indicated GSCs and DGCs, with LAMP1 serving as a lysosomal marker. f Sphere-forming frequency of GSC M83 and 456 cells with indicated modifications. sh-C, control for knock down. Vec, a control vector. WT, wide-type. Representative H&E images of mouse brain sections (g) and quantitative analysis of tumor volume (h) from athymic (BALB/c Nude) mice intracranially implanted with the GSC M83 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. i Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 4 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in three independent biological replicates. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test (a, b, d), two-sided likelihood ratio test (f), one-way ANOVA with Dunnett’s multiple comparisons test (h) or log-rank test (i). ns, not significant. Source data are provided as a Source Data file.