Journal: bioRxiv

Article Title: VISTA-induced tumor suppression by a four amino acid intracellular motif

doi: 10.1101/2025.01.05.631401

Figure Lengend Snippet: VISTA sequesters NUMB on EEA1+ vesicles. a. NUMB-GFP fusion protein was expressed in HCC1806 VISTA+ cells and co-localization was quantified for the indicated antibodies. Data represents all red signal within GFP+ vesicles from 3 independent photos at 63x resolution (N = 195, 154, 141, 176, 146 GFP+ vesicles for caveolin, EEA1, LAMP1, Rab7, Rab11, respectively). b. Confocal images of VISTA co-staining with EEA1 and Rab11. c. Co-localization of endogenous VISTA with Rab11 or EAA1 in serum starved and stimulated cells by Manders’ colocalization coefficient. N=3 pictures. d. Immunoprecipitation and immunoblotting of endogenous NUMB from HCC1806 cells +/- VISTA expression treated with serum starvation for 2 days, then stimulated (serum + EGF) for 30 minutes. e. WT and VISTA+ HCC1806 cells expressing Flag-Rab11 were subjected to Flag IP then immunoblotting. f. Quantitation of e. by densitometry. N=3 independent experiments. *p<0.02 by unpaired t-test. g. Schematic of VISTA protein sequence with sub-deletions in region 2 (2A, 2B) and sequence alignment of NPGF motif across various vertebrate species. h. 3D protein sequence alignment of crystalized PTB domains from NUMB (PDB: 5NJJ, red) and GULP1 (PDB: 6ITU, teal). i. VISTA 26-mer peptides centered on the NPGF motif were synthesized with and without mutations in NPGF and tested for interaction with NUMB-PTB proteins by streptavidin pulldown, followed by immunoblotting. j. Fluorescence polarization measurement of wt and mutant VISTA peptides binding to recombinant NUMB-PTB protein. K D calculated for wt peptide binding to NUMB-PTB. k. Immunoblot of HCC1806 cell lines engineered with CRISPR knockout of NUMB and NUMBL, and VISTA expression. l. Proliferation measurement of HCC1806 cell lines by Incucyte in the presence or absence of EGF. m. Immunoblots of cells grown without serum or EGF for 2 days, then stimulated with serum and EGF for the indicated times.

Article Snippet: Immunoblotting & immunofluorescence antibodies: α-VISTA (Cell Signaling #64953; R&D #MAB71261), α-EGFR (Cell Signaling #426 7 ), α-phospho-EGFR Y1068 (Cell Signaling #3777), α-phospho-Akt S476 (Cell Signaling #4058), α-Akt (Cell Signaling #4685), α-phospho-ERK (Cell Signaling #9101), α-ERK (Cell Signaling #4695), α-PD-L1 (Cell Signaling #13684), α-actin (Invitrogen #MA515452), α-NUMB (Cell Signaling # 2756S), α-Rab11FIP1 (Cell Signaling #12849S), α-Rab11 (Cell Signaling #5589S), α-Rab7 (Cell Signaling #9367), α-caveolin (Cell Signaling #3267), α-EEA1 (Cell Signaling # 3288), α-LAMP1 (Cell Signaling #9091).

Techniques: Staining, Immunoprecipitation, Western Blot, Expressing, Quantitation Assay, Sequencing, Synthesized, Fluorescence, Mutagenesis, Binding Assay, Recombinant, CRISPR, Knock-Out