Extended Data Fig. 4: Cdk5 knockdown promotes MHC-I re-expression in BrM cells, and it is dependent on Cdk5’s kinase activity.
A. Flow cytometry histograms showing surface expression of MHC-I in indicated cell lines. B. Mean fluorescence intensity of samples in A. C. Quantification of surface MHC-I staining intensity in cultured 4T1.Br3.shCtl and 4T1.Br3.shCdk5 #2 cells. Each dot indicates a field of view. N = 11 fields of view for 4T1.Br3.shCtl and 10 fields of view for 4T1.Br3.shCdk5 #2, pooled from at least 3 culture wells. Two-sided T-test. D. Representative MHC-I staining of 4T1.Br3.shCtl and 4T1.Br3.shCdk5#2 cells. Scale bar = 20 μm. E. mRNA expression of APP genes in 4T1.Br3.shCtl and 4T1.Br3.shCdk5#1 cells. N = 3 technical replicates. F. mRNA expression of APP genes in T11.Br1.shCtl and T11.Br1.shCdk5#2 cells. Und. – signal undetected. N = 3 technical replicates. G. Histograms showing surface expression of HLA-A/B/C in the indicated cell lines. H. mRNA expression of HLA-A/B/C genes, ERAP1, and CDK5 in the indicated cell lines. N = 3 biological replicates. Two-sided T-test. I. EO771.Br3 cells were treated with 7.5 μM RSV, harvested at indicated timepoints, and probed for indicated proteins. J. DKAT cells were transfected with an empty vector, CDK5-overexpressing vector (CDK5wt), and a vector containing dominant negative CDK5 mutation (CDK5kd), followed by probing for indicated proteins. Kinase activity of CDK5kd cells is impaired compared to CDK5wt cells. K. Flow cytometry histograms showing surface expression of MHC-I in DKAT cells transfected with vectors overexpressing wild-type CDK5 (CDK5wt), defective dominant negative CDK5 mutant (CDK5kd), or control (Vec. Ctl). L. Expression of HLA-A and HLA-B genes in MDA-BM-231.Br3 primary MFP breast tumours (N = 5 mice) and MDA-BM-231.Br3 BrM (N = 6 mice). Two-sided T-test. M. Expression of HLA-A and HLA-B genes in breast tumours and matched BrM (N = 21 patients) from the dataset of Varešlija et al. Paired two-sided T-test. N. Expression of HLA-A and HLA-B genes in breast tumours and matched BrM (N = 16 patients) from the GSE125989 dataset. Paired two-sided T-test. For L-N, results are shown as means (red line). For other panels, results are shown as means ± SEM. N.S. – not significant. BrM – brain metastasis.
