Figure 4. Absence of growth supplements induces TP53-dependent autophagy.
(A) Formation of red punctae following autophagy induction in the absence of growth supplements. HMECTERT/ST/ER-RasV12 cells (TP53+/+) or TP53 CRISPR knockout HMECTERT/ST/ER-RasV12 cells (TP53-/-) cells were transfected with the plasmid RFP-GFP-LC3. Cells were incubated in medium without EGF, insulin, and hydrocortisone for 24 hr before visualization on a confocal microscope. Scale bar: 5 μm (B) Quantification of the fraction of red punctate cells within the total population of transfected cells shown in (A). Red punctate cells were counted as cells containing only RFP signal without visible overlap of GFP signal in the cytoplasm; transfected cells were counted as cells containing either RFP signals or a mix of RFP and GFP signals in the cytoplasm (>250 cells were counted). (**p<0.01, student’s t-test) (C) Control or TP53 knockdown (TP53 shRNA) HMECTERT/ST/ER-RasV12 cells were seeded, treated and visualized as in (A). (D) Quantification of the fraction of red punctate cells within the total population of transfected cells shown in (C), treated as in (B). (**p<0.01, student’s t-test) (E) Western blot analysis of TP53+/+ cells, TP53-/- CRISPR knockout HMECTERT/ST/ER-RasV12 clones, as well as control and TP53 shRNA cells treated with or without chloroquine (CQ, 50 μM, 2 hr) after incubation in medium without EGF, insulin, and hydrocortisone for 24 hr. β-ACTIN serves as the loading control. (F) Western blot analysis of HMECTERT/ST/ER-RasV12 cells and two independent TP53 CRISPR knockout HMECTERT/ST/ER-RasV12 clones. Cells were incubated in medium in the presence or absence of EGF, insulin, and hydrocortisone (denoted as EIH) for 48 hr. β-ACTIN serves as the loading control. (G) qRT-PCR analysis of cells in (F). TGM2 mRNA expression was normalized to TBP mRNA expression. The mean value of TGM2 mRNA expression in TP53+/+ cells with presence of EIH is set at 1, and relative expression is shown. (**p<0.01, ns: not significant, student’s t-test) (H) Western blot analysis of HMECTERT/ST/ER-RasV12 cells expressing either control or TP53 shRNA. Cells were treated the same as in (F). (I) qRT-PCR analysis of cells in (H). Data are shown as in (G).
Figure 4—figure supplement 1. DNA sequencing of TP53-/- clones.
