Extended Data Fig. 3: p62/SQSTM1 is required for the effects of NBR1 overexpression on tumour metastasis and pro-metastatic basal differentiation.
From: Autophagy-targeted NBR1–p62/SQSTM1 complexes promote breast cancer metastasis by sequestering ITCH
a, Immunofluorescent staining for p62 (green) and CK5 (red) in MDA-MB-231 cells expressing indicated shRNAs and, when indicated, treated with Ulk-i. Offset: magnification of boxed area in Ulk-i+shCTRL cells. b, Immunoblot analysis of MDA-MB-231 and R221A cells from indicated cohorts. Loading control (actin) corresponds to same blot. c, Immunofluorescent staining of indicated MDA-MB-231 cell cohorts for FLAG (NBR1, green), p62 (red) and nuclei (DAPI, blue). d, Quantification of total number of metastases per section of lung for shCTRL (n = 7), WT-NBR1 (n = 6), WT-NBR1 + shp62, #1 (n = 8), wt-NBR1 + shp62, #2 (n = 9). e, Representative H&E staining of lung metastases from indicated groups. f,g, Lungs bearing metastases from indicated groups stained for p63 (green, f) or CK14 (green, g) and nuclei (DAPI, blue). h, PyMT R221A cells expressing the indicated constructs stained for p63 (green, left panel) and CK14 (green, right panel) and nuclei (DAPI, blue). i, Quantification of p63 (nuclear staining) and CK14 positive R221A cells. 400 or more cells from each of three individual experiments were counted per condition. j, Quantification of total number of metastases per section of lung in shCTRL (n = 12), WT-NBR1 (n = 11), D50R-NBR1 (n = 10). shCTRL and WT-NBR1 cohorts were also included as controls in d and Extended Data Fig. 7a. k, Lungs bearing metastases from the indicated groups stained for p63 (green, top), CK14 (green, bottom) and DAPI (blue). l, Quantification of p63 (nuclei) and CK14 positive cells within lung metastases for the indicated conditions. Minimum of 250 cells from multiple (4–8) metastases counted per cohort. A total of five (n = 5) individual mice from three independent experiments evaluated. Statistical significance determined by either one-way (d,j,l) or two-way (i) ANOVA, followed by Dunnett’s multiple comparisons test. Data are represented by mean ± S.E.M; each dot represents one animal (d,j,l) or an individual experiment (i). Data and unprocessed blots are provided.
