Fig. 2: CD44 negatively regulates autophagy.

a, b Western blot analysis of LC3B-II/LC3B-I and SQSTM1 (a) or immunohistochemical staining for SQSTM1 (b) of aortas obtained from WT or CD44-KO mice (16–19 months) treated with vehicle or chloroquine (CQ) by daily intraperitoneal injection (60 mg/kg/day) for 4 days. Bar = 200 μm. Bar in zoomed figure = 50 μm. c, g Fluorescence analysis of GFP-LC3B puncta in HUVECs (PD3-6) transfected with scrambled siRNA or CD44 siRNAs (c), or transduced with Ev or CD44ICD (g) in the presence or absence of bafilomycin A₁ (100 nM) for 6 h. d, h Western blot analysis of LC3B-II/LC3B-I and SQSTM1 in HUVECs (PD3-6) transfected with scrambled siRNA or CD44 siRNAs (d), or transduced with Ev or CD44 (h) in the presence or absence of bafilomycin A₁ (100 nM) for 6 h. e, j Fluorescence analysis of CD44 knockdown (e) or CD44ICD-overxpressing (j) HUVECs (PD3-PD6) transfected with mCherry-GFP-LC3B reporter. HUVECs treated with bafilomycin A₁ (100 nM) for 6 h were used as a positive control for impaired autophagic flux. HUVECs incubated with EBSS for 3 h or treated with rapamycin (100 nM) for 6 h were used as positive controls for enhanced autophagy activity. Nuclei were stained with DAPI. Quantification of the average number of autolysosomes (GFP⁻mCherry⁺ puncta, red) and autophagosomes (GFP⁺mCherry⁺ puncta, yellow) per cell. Bar = 10 μm. f, i Ultrastructural analysis of HUVECs (PD3-6) transfected with scrambled siRNA or CD44 siRNAs (f), or transduced with Ev or CD44 (i). Bar = 500 nm. Boxed areas are enlarged below each image. For a, n = 6 (left bar graph) or 7 (right bar graph) mice per group. For b, n = 5 mice per group. Two (a, b) or three (c–g, j) or four (h) biologically independent experiments. Data are shown as mean ± s.d.; P values are derived from Two-tailed unpaired Student’s t-tests (a, b), one-way ANOVA with Dunnett’s multiple comparisons test and Two-tailed unpaired Student’s t-tests (c–e, g, h, j). Source data are provided as a Source data file.