Fig. 5.

NVP-AUY922 induces IGF-1Rβ degradation partially via a chaperone-mediated autophagic pathway. a, b The mRNA levels of HSC70, LAMP2A and LAMP1 were detected in Mia-paca2 and Capan-2 cells after incubation with the indicated concentrations of 922 for 24 h by qPCR. GAPDH served as an internal standard. c, d The LAMP2A and HSC/HSP70 expression levels in Mia-paca2 and Capan-2 cells after treatment with the indicated doses of 922 for 24 h were analyzed by western blots. β-actin served as a loading control. e, f LAMP2A or IGF-1Rβ coIP assays. Lysates (500 μg) from Mia-paca2 cells treated with 922 (1 μM) for 6 or 24 h were immunoprecipitated with LAMP2A or IGF-1Rβ antibodies, and the precipitates were used for western blot assays to evaluate LAMP2A, HSC70/HSC70 or IGF-1Rβ protein levels. IgG served as an internal control. Input: total cell lysate. g, h After 24 h of 922 (1 μM) treatments, Mia-paca2 and Capan-2 cells were stained with anti-LAMP2A (green) and anti-HSC/HSP70 (red) antibodies and then visualized by confocal fluorescence microscopy. DAPI (4′,6-diamidino-2-phenylindole) was used to label the nucleus (blue). Representative images are presented (400 ×). i The LAMP2A mRNA level after LAMP2A knockdown by vector-mediated RNAi. j Silencing of LAMP2A for 24 h in Mia-paca2 cells and then treated with 1 μM of 922 for another 24 h. The IGF-1Rβ and LAMP2A expression levels were analyzed by western blot. The data are expressed as the mean ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.01 versus con