Fig 2: RNA-seq results were validated by Western blot analysis in WT and calpain-1 KO mice. Western blot images and quantifications showing the expression levels of HSPA1B, DNAJB1, IDE, ARC, PER2, PLA2G4E, and NR4A1 proteins in three independent replicates, respectively. Dot plots were used for the quantification of the expression levels of 7 proteins compared to actin control in WT and calpain-1 KO mice. Unpaired t-test in Prism 7 was used to calculate p values, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Identification of endothelial cells in TNBC biopsies. (a) Flow cytometry analysis revealed three endothelial cell subclusters in TNBC biopsied tissues, featured by the expression of ATP1B3, HSPA1B, and KRT7, respectively. Data represent similar results acquired from five independent experiments. (b) UMAP plots showed 9 cell types in the normal human breast and 10 cell types in TNBC biopsy tissues. The boxed cell types indicate endothelial cells. (c) Violin plots exhibited the expression pattern of endothelial cell markers (CD93, CDH5, and PECAM1) and a leukocyte marker (PTPRC). (d) Flow cytometry analysis identified the expression of CDH5 and CD93 from endothelial cells in TNBC biopsies. Data represent similar results acquired from three independent experiments.

Fig 4: Heat maps showing temporal expression of statistically significantly (p < 0.05) expressed genes at conclusion of heat stress (0H) and during recovery at 37 °C at 1 h (1H), 3 h (3H), 6 h (6H), and 24 h (24H). Maximal gene-expression time point at 1H in DBMSCs (A), 3–6H in DPMSCs (B), and 3H in pMSCs (C). Additionally, in all the three cell types, significantly high expression of HSPA1A, HSPA1B, DNAJA4, and DNAJB1at 0H is visible. (For Fold Change values and Column Representation, see Table S2 and Figure S2).

Fig 5: Dose-responsive nature of heat stress in DBMSCs (A) Time-course analysis of Hsp70 gene expression induction at 42 °C for 1 and 2 h and at 44 °C for 1, 2, and 3 h. (B) Log2 Fold Change plot for Hsp70 induction. Temperature elicits larger response as can be judged from difference between 44-2H and 44-1H. As the magnitude of heat stress increases, the heat shock response increases both in magnitude and duration as seen at 9H and 12H for 44-2H and 44-3H. (C) Dose responsiveness shows a pattern in statistical significance as the p-value varies decreases with respect to increase in magnitude of heat stress from 42-1H to 44-3H. It is further evident from p < 0.05 in the case of 44-2H and 44-3H up to 12 h post-heat stress.