Journal: Journal of Translational Medicine

Article Title: Caveolin-1 drives immunosuppression in esophageal squamous cell carcinoma by enhancing exosome secretion and macrophage M2 polarization via inhibition of MVB autophagic degradation

doi: 10.1186/s12967-026-07800-3

Figure Lengend Snippet: CAV1-KD induces autophagy-mediated MVB degradation and reduces M2 polarization (A) WB of EEA1, LC3B-II/I, and p62 in control and CAV1-KD cells ( n = 3 per group). β-actin used for normalization. (B–D) Representative TEM images showing MVB and ILV morphology and abundance in control and CAV1-KD cells ( n = 3 per group). Green arrows indicate MVBs; Scale bar = 200 nm. (E–G) Representative TEM images showing AV abundance and MVB–AV fusion events in control and CAV1-KD cells ( n = 3 per group). Enlarged image of CAV1-Ctrl shows normal MVB structure, while those of CAV1-KD1 and CAV1-KD2 cells show MVBs fused with AVs. Red arrows indicate AVs; Scale bar = 1 μm. (H) Representative IF images of MVB and AV localization in CAV1-Ctrl, Rapa-treated CAV1-Ctrl, CAV1-KD, and 3-MA-treated CAV1-KD cells ( n = 3 per group); Scale bar = 10 μm. Colocalization analysis was performed using ImageJ. (I) NTA analysis of exosome particle concentration ( n = 3 per group). (J) BCA assay for exosomal protein content ( n = 3 per group). (K–M) qRT-PCR of M2 markers in macrophages treated with exosomes ( n = 3 per group). (N) WB of CAV1, EEA1, LC3B-II/I, and p62 ( n = 3 per group). β-actin used for normalization. (O) WB of PI3K, AKT, mTOR, and phosphorylated forms in control and CAV1-KD cells ( n = 3 per group). β-actin used for normalization. Data are presented as mean ± SD or median and analyzed by one-way ANOVA followed by Tukey’s post hoc test. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Following blocking with 5% non-fat milk (BD Difco, #232100, USA) or a commercial blocking solution (Yoche, #YWB0501, China), membranes were incubated overnight at 4 °C with primary antibodies against Calnexin (Proteintech, #66903-1-Ig; 1:10,000), CD9 (Proteintech, #60232-1-Ig; 1:30,000), CD63 (Proteintech, #67605-1-Ig; 1:12,000), TSG101 (Proteintech, #67381-1-Ig; 1:40,000), CAV1 (HUABIO, #ET1603-1; 1:2000), RAB34 (ABclonal, #A10332; 1:2000), TSPAN1 (Proteintech, #16058-1-AP; 1:500), EEA1 (ABclonal, #A0592; 1:1000), LC3B (ABclonal, #A19665; 1:2000), p62 (Selleck, #F0106; 1:10000), p-PI3K (Abmart, #TA3241; 1:500), PI3K (Abmart, # T40115 ; 1:1000), p-Akt (Abmart, # T40067 ; 1:1000), pan-Akt (Selleck, #F0004; 1:1000), p-mTOR (Selleck, #F2584; 1:5000), mTOR (Selleck, #F0169; 1:1000), p-AMPK (Selleck, #F0151; 1:1000), AMPK (Selleck, #F0269; 1:1000), and β-actin (Proteintech, #66009-1-Ig; 1:50,000).

Techniques: Control, Concentration Assay, BIA-KA, Quantitative RT-PCR