Fig. 3

Autophagy promotes the stemness of M14-SE cells. a Western blot analysis for LC3I/II and P62 in M14-Parental and M14-SE cells. b M14-Parental and M14-SE cells expressed mRFP-LC3B fusion protein via adenovirus infection. Representatives of LC3B-positive puncta images were shown, bar = 20 μm. c Transmission electron microscopy of M14-Parental and M14-SE cells. Autolysosomes indicated by arrowheads, bar = 5 μm. d Mean number of detectable autolysosomes in each tumor cell, counted on transmission electron microscopy images. e–g Clonogenic spheroid formation rate, morphology and average spheroid diameter of M14-SE cells treated with DMSO, 3-MA, Baf-A1 and Rapa in the single-cell cloning assay. h and i RT-qPCR and Western blot analysis to confirm effective Atg5 interference in M14-SE cells. j Analysis of mRNA expression of Aldh1, CD133, Nanog, Oct4 and Sox2 in M14-SE cells upon Atg5 interference. k–m Clonogenic spheroid formation rate, morphology and average spheroid diameter of M14-SE cells with Atg5 interference in the single-cell cloning assay. n Serial spheroid formation assay of M14-SE cells with Atg5 interference, bar = 200 µm. o Tumors resected from nude mice inoculated with M14-SE cells with Atg5 interference, bar = 1 cm. p and q Tumor volume and weight, *P < 0.05, **P < 0.01, ***P < 0.001