Extended Data Fig. 4: Autophagy inhibition promotes transcription of p63 target genes and p63 protein stability.
From: Autophagy-targeted NBR1–p62/SQSTM1 complexes promote breast cancer metastasis by sequestering ITCH
a, qRT-PCR analysis of p63 and p63 target mRNA expression in mouse 4T1 cells for the indicated groups. b,c, qRT-PCR analysis of p63 mRNA expression in human triple-negative MDA-MB-231 cells (b) and mouse PyMT R221A (c) cells for the indicated groups. d, Quantification of p63 protein levels in MDA-MB-231 cells following cycloheximide treatment. Following ATG7 knockdown or 18 h treatment with indicated agents, cells were treated with cycloheximide for indicated times, lysed, subject to immunoblot analysis and integrated band intensity was calculated via densitometry. For each condition and timepoint, integrated band intensity of p63 protein was normalized to both its respective actin control and the corresponding t = 0 timepoint. e, Representative immunoblot from cycloheximide chase assays of MDA-MB-231 cells used for quantification in d. MG132 employed as a control to inhibit proteosomal degradation of p63. Samples were run on separate gels with equal loading of total proteins. f, Representative immunofluorescent staining of CK14 (red) and nuclei (DAPI, blue) in lung metastasis from indicated groups. g, Phase-contrast micrograph of human MDA-MB-231 and mouse PyMT R221A cells 24 h following treatment with CTRL or p63 shRNAs. h, Working model: autophagy inhibition in breast cancer cells results in accumulation of the ACRs NBR1 and p62/SQSTM1, which in turn, increases protein levels of the p63 transcription factor and enhanced expression of p63-target genes. Data (a–c) are represented by mean ± S.E.M. Each dot represents a technical replicate within one bio-replicate; one representative of three bio-replicates are shown. Each data point in d represents mean band intensity ± S.E.M from three (n = 3) independent experiments. Data and unprocessed blots are provided.
