crispr cas9 knockout  (Addgene inc)

Journal: Journal of Hematology & Oncology

Article Title: CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies

doi: 10.1186/s13045-025-01774-3

Figure Lengend Snippet: Targeting CD47 with SRF231 induces phagocytosis and cell death in lymphoid malignant cells. A. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in Jurkat cells cocultured with hMDM ( n = 2). B. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in 11 CLL patient samples cocultured with hMDMs from 3 different donors (MAC1—MAC3). Reported P values were calculated by paired Student’s t test. C. Diagram showing gating strategy – Phagocytosis is identified as the CD14 + population with uptake of CFSE + lymphocytes. SRF231-mediated cell death is identified as the non-phagocytosed CD14 − cells with CFSE and Violet Live/Dead uptake. Violet Live/Dead (Zombie Violet™) is an amine-reactive fluorescent dye that stains cells that have compromised membranes as compared to AnnV stain that binds phosphatidylserine. Representative flow cytometry analyses showing the effect of hIgG4 isotype (10 µg/ml, top panel) and SRF231 (10 µg/ml, bottom panel) on phagocytosis and cell death of non-phagocytosed cells, cocultured with hMDMs. D. Population of non-phagocytosed Jurkat cells following treatment with either hIgG4 isotype or SRF231 were analyzed by flow cytometry. Tumor cell death denoted by % of death of non-phagocytosed cell population ( n = 2). E . Cells from 24 CLL patient samples were subjected to Protein G-bound SRF231 (10 µg/ml) and cell viability were measured by AnnV/Hoechst cell death assay. Decrease in percent live (AnnV-/Hoechst+) cells in SRF231 vs. hIgG4 isotype treatment was reported. Reported P values were calculated by paired Student’s t test. F. Healthy B cells (HB, n = 8), healthy T cells (HT, n = 8), healthy monocytes (HM, n = 3) and PBMC cells from CLL patients (CLL, n = 24) were subjected to Protein G-bound SRF231 or hIgG4 isotype treatment (10 µg/ml). Cell death induction was measured using AnnV/Hoechst assay at 0, 2, 4, 6 and 10 h, and data presented were % live cells of SRF231 normalized to their respective hIgG4 isotype. Reported P values were calculated by Sidak’s multiple comparison test. G-H. Western Blot showing CD47 and β-actin protein expressions in primary HB and CLL cells, Ri-1, Raji and Jurkat (Wild type and CD47 Knockout) cells. I. Cell death inductions of Ri-1 ( n = 3, 6 h; n = 4, 24 h), Jurkat ( n = 4) and Raji ( n = 4) cells were measured following Protein G-bound SRF231 incubation with AnnV/Hoechst assay at indicated timepoints. Reported P values were calculated by paired Student’s t test

Article Snippet: Ri-1 (Sigma-Aldrich, cat# 96090512), Raji (ATCC, cat# CCL-86), Jurkat (Clone E6-1, ATCC, cat# TIB-152) and Jurkat CD47 CRISPR/Cas9-knockout (KO) (Applied StemCell), OCI-Ly3 (DSMZ, cat# ACC761), OCI-Ly1, OCI-Ly1-R, TMD8 (Wu Lab, DFCI), MOLM14-S (Chng Lab, CSI, NUS), MOLM14-R, HL60 (Pervaiz Lab, NUS) cell lines were maintained in R10.

Techniques: Staining, Flow Cytometry, Comparison, Western Blot, Knock-Out, Incubation

Journal: Journal of Hematology & Oncology

Article Title: CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies

doi: 10.1186/s13045-025-01774-3

Figure Lengend Snippet: SRF231 induces cell death through necroptotic pathway. A . Schematic illustration of necroptosis signalling activation involving the phosphorylation of key proteins such as RIPK1/RIPK3 and MLKL as well as their respective inhibitors. Created in BioRender. Chamberlain, S. (2026) https://BioRender.com/toqddow . B-C. Western Blot showing the increased phosphorylation of RIPK1 (S166) and MLKL (S358) after Protein G-bound SRF231 treatment of Ri-1 cells (10 µg/ml, 24 h) or primary CLL cells (10 µg/ml, 6 h). D . Images of 6-hour hIgG4 isotype and SRF231 treated primary CLL cells displaying the intensity of p-RIPK1 and p-MLKL (all green), DAPI (blue) and merge using confocal microscopy (60x). E. Western blot showing the p-MLKL of wild-type Jurkat and CD47-KO Jurkat cells following treatment with Protein G-bound hIgG or SRF231 (10 µg/ml, 24 h). F. Cell viability of Jurkat cells treated with Protein G-bound hIgG or SRF231 (10 µg/ml, 24 h) following 48-hour siMLKL was measured with AnnV/Hoechst assay ( n = 4). Reported P values were calculated by Sidak’s multiple comparison test. G. Effect of 1-hour pre-treatment with RIPK3 inhibitor (GSK’872, 50 nM) or RIPK1 inhibitor (Necrostatin-1, 1 µM) on 6-hour Protein G-bound SRF231-induced cell death in 14 patient samples, was measured by AnnV/Hoechst assay. Reported P values were calculated by paired two-tailed Student’s t test. H. Western blot showing the effect of 1-hour pre-treatment with RIPK3 inhibitor (GSK’872, 50 nM) or RIPK1 inhibitor (NEC-1, 1 µM) followed by 6-hour treatment with Protein G-bound SRF231 (10 µg/ml) on p-MLKL in primary CLL cells. Total ERK and PGAM5 were used as loading controls. I. p-RIP1K and p-MLKL immunohistochemistry staining of hIgG control- or SRF231-treated formalin-fixed paraffin embedded tissues from subcutaneously implanted Raji tumors in CB17.SCID mice. J. Quantification of 6 representative region of interests (ROI) per sample of p-RIP1K and p-MLKL IHC staining in Fig. 2I. ( n = 3 per treatment). Reported P values were calculated by unpaired two-tailed Welch’s t test. OD: Optical Density

Article Snippet: Ri-1 (Sigma-Aldrich, cat# 96090512), Raji (ATCC, cat# CCL-86), Jurkat (Clone E6-1, ATCC, cat# TIB-152) and Jurkat CD47 CRISPR/Cas9-knockout (KO) (Applied StemCell), OCI-Ly3 (DSMZ, cat# ACC761), OCI-Ly1, OCI-Ly1-R, TMD8 (Wu Lab, DFCI), MOLM14-S (Chng Lab, CSI, NUS), MOLM14-R, HL60 (Pervaiz Lab, NUS) cell lines were maintained in R10.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Confocal Microscopy, Comparison, Two Tailed Test, Immunohistochemistry, Staining, Control, Formalin-fixed Paraffin-Embedded