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. 2023 Oct 16;42(47):3529–3541. doi: 10.1038/s41388-023-02864-7

Fig. 5. Primary cilia formation and autophagic flux in human osteosarcoma in vitro.

Fig. 5

a Immunofluorescence colocalization of acetylated α-tubulin (AcTUB) and ARL13B in human osteosarcoma cell lines cultured in 10% serum or serum-free media for 24 h. Representative images are shown, along with corresponding genotypes for each cell line. Scale bar = 5 µm. b Quantification of primary cilia frequency in human osteosarcoma cell lines cultured in 10% serum or serum-free media for 24 h. n = 5 individual experiments, mean + SEM, **P < 0.01, ***P < 0.001, Student’s unpaired t test. c Confocal immunofluorescence images of primary cilia stained for AcTUB and SMO in B143 human osteosarcoma cells treated with PBS, SHH (rhSHH) and/or sonidegib (LDE225). Scale bar = 5 µm. d Quantification of primary cilia co-expressing SMO in B143 human osteosarcoma cells treated with treated with PBS, SHH (rhSHH) and/or sonidegib, n = 11–14 ciliated cells per treatement. e Western blot analysis of LC3 and ACTIN expression in non-ciliated (U2OS) and ciliated (MG63) human osteosarcoma cell lines cultured in 10% serum or serum-free with or without 50 nM bafilomycin-A (Baf-A) for 24 h. n = 5 individual cell lines. f Quantification of autophagic flux by western blot in non-ciliated (U2OS) and ciliated (MG63) human osteosarcoma cell lines as shown in (e). n = 5 independent experiments, mean ± SEM. Graph represents fold change in autophagic flux in serum-free media (induced autophagy) compared to normal serum (basal autophagy) *P < 0.05, Student’s unpaired t test.