Fig 2: Src family kinases for BLK, LCK, and LYN interaction with TRPM8.A GST pull-down assays to assess the interaction between purified GST-tagged C-terminus of TRPM8 (GST-M8C) expressing in E. coli BL21 bacteria and different HA-tagged Src family kinases expressing in HEK293T cells. Western blotting (WB) was performed using the indicated antibodies. B–D Co-immunoprecipitation (Co-IP) assays. B HeLa cells were transfected with the indicated constructs along with Flag-tagged full-length TRPM8 (Flag-TRPM8). Immunoprecipitation (IP) was performed with an anti-HA or anti-Flag antibody, and the samples were analyzed by immunoblotting with the indicated antibodies. C Similar Co-IP in (B) but with protein extracts from HeLa cells co-transfected with the indicated constructs along with GFP-tagged C-terminus of TRPM8 (GFP-M8C). D Co-IP as in (B) and (C) but with protein extracts from native PANC-1 cells. E Representative confocal imaging of co-localization of mcherry-LCK and GFP-TRPM8 in HeLa cells. Overlay images show co-localization of green signals (TRPM8) and red signals (LCK), which generated yellow signals in HeLa. Nuclei were stained with DAPI (blue). Scale bars, 10 µm. F Assay of the interaction in vitro between purified His-LCK fusion and GST-tagged C-terminus of TRPM8 (GST-M8C) from E. coli bacteria. G, H HEK293T cells co-expressing HA-LCK constructs with a series of mutant Flag-tagged cytoplasmic domain of TRPM8 were harvested for Co-IP assays. All studies were repeated at least three times. GFP, green fluorescent protein. All studies were repeated at least three times.

Fig 3: BCR pathway perturbations drive changes in cell mass and stiffness in Jeko-1 cells.(A) Schematic illustrating BCR inhibition via acalabrutinib treatment (top panel), BCR stimulation through anti-IgM treatment (middle panel) and BCR pathway enhancement through BLK and CD79A overexpression (bottom panel) in Jeko-1 wild type (wt) cells for biophysical profiling. BCR inhibition was confirmed by western blot, and stable overexpression in cell lines was validated using RT-PCR, western blot, and flow cytometry. Functional assays (cell cycle and proliferation) further assessed GFP, BLK or CD79A-expressing cells. Created with BioRender. (B) Median single-cell mass and stiffness measurements obtained using the SMR from >500 Jeko-1 wild type cells after 1h or 24h of ex vivo treatment with DMSO, 0.25 μM, or 0.5 μM acalabrutinib. Data are presented as the median ± SD of three biological replicates. (C) Median single-cell mass and stiffness measurements from >500 Jeko-1 wt cells, with or without IgM stimulation for 10min or 24h, assessed using the SMR. Data represent the median ± SD of three biological replicates. (D) Relative mRNA expression of BLK and CD79A measured by RT-qPCR in Jeko-1 overexpressing GFP, BLK, or CD79A. Results are presented as fold change normalized to GAPDH mRNA. Each bar represents the mean ± SEM of two independent stable cell lines. (E) Representative images of western blot showing BLK, CD79A, GFP and α-actinin protein in Jeko-1 overexpressing GFP, BLK or CD79A. (F) Median single-cell mass and stiffness measurements obtained using the SMR from >500 cells of Jeko-1 wild type (WT) and Jeko1 cells stably overexpressing GFP, BLK or CD79A. Data are presented as median ± SD of three biological replicates. NS: non-significant, *P < 0.05, **P < 0.01 as compared between indicated groups (Student’s t-test).