Fig 2: PLC-gamma-2 antibody screening by Western Blot.Lysates of THP-1 (WT and PLCG2 KO) either treated (+) or not treated (-) with PMA were prepared and 10 μg of protein were processed for Western Blot with the indicated PLC-gamma-2 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. All antibodies were tested at a dilution of 1/500 except antibody 34264** was tested at 1/200. Predicted band size: 148 kDa. *Monoclonal antibody, **Recombinant antibody.

Fig 3: PLC-gamma-2 antibody screening by immunoprecipitation.THP-1 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated PLC-gamma-2 antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for Western Blot with the indicated PLC-gamma-2 antibody. For Western Blot, 55512** was used at 1/500. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.

Fig 4: Identification of EMT cells. (a) Cell density map of cell clusters across photostimulation and control groups. (b) Proportional distribution of cell numbers across subpopulations in each group. (c) Top 10 genes with the highest fold-change differences in expression between Cluster 2 and other subpopulations. (d) Violin plots showing the expression levels of PLCG2 across all clusters. (e) Feature plot of PLCG2 expression across all clusters. (f) Experimental timeline: tumors inoculated on day 0, photostimulated on day 7, and harvested on day 10 for immunofluorescence analysis. (g) Representative immunofluorescence images of PLCG2 (green) in cells on day 1. Blue: DAPI. Scale bar: 100 μm. Right: Quantification of PLCG2 levels (control and laser: n = 4 fields × 3 cell dishes per group). Statistical significance was determined using a two-tailed t-test. Data represent mean ± SEM. * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. (h) Representative immunofluorescence images of PLCG2 (red) in tumor tissues. Blue: DAPI. The images in second row show magnified views of the images in first row with nuclear outlines highlighted by dashed lines. Scale bar: 20 μm for the first row; 5 μm for the second row.

Fig 5: PLCG2 inhibitor suppresses subcutaneous tumor development in mice without affecting metastases. (a) Representative images of Ca2+ dynamics before and after photostimulation in the presence of U73122. Green fluorescence: Fluo-4/AM. Scale bar: 50 μm. Kinetic plots of Ca2+ by photostimulation at 10.5 mW (laser + U73122 n = 54 cells, laser n = 45 cells). (b)–(d) Immunofluorescence of E-cadherin (b), N-cadherin (c), and Vimentin (d) after laser stimulation in the presence of U73122. Corresponding quantified expression levels are shown for E-cadherin and N-cadherin (control and laser: n = 3 fields × 3 cell dishes per group) and for Vimentin (control and laser: n = 4 fields × 3 cell dishes per group). Statistical significance was determined using a two-tailed t-test. Data represent mean ± SEM. * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. Scale bar: 50 μm. (e) Experimental timeline: tumors inoculated on day 0. From day 22 to day 35: U73122 treatment (30 mg/kg, 3 mg/ml). Group 1: immunofluorescence microscopy of subcutaneous tumors on day 35; group 2: in vivo imaging on day 70. (f) Weight curves of group 1 mice during the treatment period (U73122: n = 5, Control: n = 6). (g) Tumor volume of group 1 mice during the treatment period (U73122: n = 5, Control: n = 6). (h) Representative immunofluorescence images of PLCG2 (red) and FN1 (red) in tumor tissues from group 1 on day 35. Blue: DAPI. Upper: wide-field images (scale bar: 150 μm). Lower: magnified views of selected regions (scale bar: 50 μm). (i) Weight curves of group 2 mice during the total period (U73122: n = 5, Control: n = 5). (j) Tumor volume curves of group 2 mice during the total period (U73122: n = 5, Control: n = 5). (k) Representative in vivo bioluminescence imaging using luciferase to monitor metastases in group 2 mice. Green arrows: primary tumors; red arrows: metastases. Right: Quantitative analysis of metastases (U73122: n = 5, Control: n = 5). Data represent mean ± SEM.