Human TP53INP1 activates autophagy.

<p>Panel A. HeLa cells were transiently transfected with TP53INP1 and incubated with DMEM (basal) or HBSS for 1 h (amino acid starvation), or 2 µM rapamycin for 3 h. The intracellular localization of TP53INP1 was analyzed by immunofluorescence and is shown in red. The nuclei are shown in blue. Scale bars, 10 µm. Panel B. Confocal images of HeLa cells transiently transfected with TP53INP1 and GFP-LC3, and incubated whether with DMEM, HBSS for 1 h or 2 µM rapamycin for 3 h. The intracellular localization of TP53INP1 was analyzed as in panel A. Scale bars, 10 µm. Contrast-corrected merged RGB pictures and Z-projection of PDM images are shown for all panels. Color scales of PDM images have different maximal values so care must be taken when comparing conditions. PDM values closer to 1 show reliable co-localized pixels. Panel C. HeLa cells were transiently transfected with either pcDNA3, DOR or TP53INP1a and incubated with DMEM or HBSS for 1 h, alone or in combination with 200 nM bafilomycin A1 for 16 h (cells were incubated with BafA1 for 16 h and for the last hour they were incubated with or without HBSS). Cell lysates were obtained and Western blot assays were performed with specific antibodies against L C3 and beta-actin. Panel D. HeLa cells previously infected with lentiviruses encoding scramble RNA or TP53INP1 shRNA were cultured. Cell extracts and total RNA were obtained and TP53INP1 mRNA levels were assayed by real-time PCR. Data are mean ± SEM of 3 independent experiments. * significant difference compared to the Scramble group, at P<0.0001. Panels E and F. Scramble or TP53INP1 shRNA HeLa cells were cultured and incubated with DMEM or HBSS for 1 h in combination with 200 nM bafilomycin A1 for 16 h (cells were incubated with BafA1 for 16 h and for the last hour they were incubated with or without HBSS). Endogenous LC3 and beta-actin were detected by Western blot (panel E). Quantification of LC3II levels relative to beta-actin was performed (panel F). Data are mean ± SEM of 3 independent experiments. * significant effects caused by TP53INP1 knockdown, at P<0.05.