Extended Data Fig. 10: Validation of Y chromosome loss in MB49 tumor models and immune cells.
From: Concurrent loss of the Y chromosome in cancer and T cells impacts outcome
a-b. Genomic DNA (10 ng per reaction) was isolated from sorted epithelial cells, non–T cells, and T cells derived from CRISPR‐engineered (Y-Scr or Y-KO) MB49 tumors. Wild‐type (WT) C57Bl/6 N genomic DNA (lacking Cas9) served as the reference in the ΔΔCt analysis. (a) Ct values were normalized to Gapdh and plotted as fold change relative to WT. (b) Ct values were normalized to Actb and similarly plotted as fold change over WT. Error bars represent mean ± s.e.m. from 4 biological replicates in each group. c. DNA qPCR analysis of Y chromosome genes in TdTomato+ CD45- epithelial cells (left) and CD3+ T cells (right) from CRISPR Y-Scr and CRISPR Y-KO groups. Fold changes normalized to Gapdh (purple dots) and Actb (yellow dots). P-values from two-way ANOVA with Fisher’s LSD test and Geisser-Greenhouse correction. Error bars represent mean ± s.e.m.; Each dot denotes pooled samples from six independent tumors. Six independent tumors were mashed and pooled together for substantial number of T cells isolation and then randomly aliquoted to 4 tubes to increase the efficacy of the result and reduce human error. Each dot of one color represent data from one such tube. d. DNA qPCR analysis of Y chromosome genes (Kdm5d, Uty, Eif2s3y, and Ddx3y) in PBMCs extracted from BBN-treated mice. Each dot represents the pooled DNA sample from 3-4 mice. On X axis: the time points of samples collection, along with a timeline of BBN administration and bladder tumor onset; on the Y axis the fold change expressed as relative gene expression level compared to untreated ctrl at initiation of BBN. e. Changes in DNA levels of Y chromosome genes (Ddx3y, Kdm5d, Zfy1/2, Ssty1, Ssty2) upon long term activation of primary mouse T cells in vitro. qPCR data normalized to housekeeping genes Actin (left) or B2m (right) relative to day 1 after mouse T cell isolation. Mean ± SD, n = 4. SD, Standard Deviation. f-g. Expression changes of T cell exhaustion markers (Ctla4, Pdcd1, Tcf7, Tim3) during long term activation of primary mouse T cells in vitro. Expression normalized to housekeeping genes Actin (f) or Gapdh (g) are shown relative to day 1 post T cell isolation. Mean ± SD, n = 4. h-i. Mean expression of exhaustion (h) and Y chromosome genes (i) in Naïve P14 CD8+ T cells at baseline (Day 0) and compared to at Days 8, 15, and 30 post-infection under acute (LCMV Armstrong; solid lines) or chronic (LCMV Clone 13; dashed lines) conditions. Data were extracted from publicly available transcriptomic datasets39.
