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

Journal: Materials Today Bio

Article Title: Targeted biomimetic fusogenic liposome enhances tumor accumulation, penetration, and therapeutic efficacy of siRNA therapeutics

doi: 10.1016/j.mtbio.2025.102414

Figure Lengend Snippet: Antitumor effects of CRPIL/siCD47 complexes. (a) Expression of CD4 7 mRNA in 4T1 cells after different treatments. (b) Schematic illustration of in vitro phagocytosis assay. (c–d) Fluorescence microscope images and quantification of phagocytosis efficiency of J774A.1 cells against 4T1 cells. (e) Schematic illustration of in vitro tumor cell inhibition assay. (f) Cellular viability of 4T1 cells following various treatments in co-culture with J774A.1 macrophages. (g) Schematic illustration of siRNA treatment schedule. (h) Changes in tumor volumes during the treatment. (i–j) Dissected tumors and their average weights after treatment. (k) CD47 protein expression in tumor tissues across treatment groups. (l) Infiltration of CD86 + M1 macrophages in tumors following different treatments.

Article Snippet: After washing, the membranes were incubated with CD47 Polyclonal antibody (Proteintech Group, 20305-1-AP), PLK1 Polyclonal antibody (Proteintech Group, 10305-1-AP) or GAPDH Monoclonal antibody (Proteintech Group, 60004-1-Ig) for 1 h at room temperature.

Techniques: Expressing, In Vitro, Phagocytosis Assay, Fluorescence, Microscopy, Inhibition, Co-Culture Assay

Journal: Materials Today Bio

Article Title: Targeted biomimetic fusogenic liposome enhances tumor accumulation, penetration, and therapeutic efficacy of siRNA therapeutics

doi: 10.1016/j.mtbio.2025.102414

Figure Lengend Snippet: Antitumor effects of CRPIL/siCD47/siPLK1 complexes. (a). Expression of CD4 7 mRNA and PLK1 mRNA in 4T1 cells after different treatments. (b) The phagocytosis efficiency of J774A.1 cells against 4T1 cells after different treatments. (c) Schematic illustration of treatment schedule. (d) Changes in tumor volumes during the treatment. (e–f) Photographs of dissected tumors and average tumor weights at the end of the treatment. (g) Expression of CD47 and PLK1 proteins in tumor tissues after different treatments. (h) Representative images of hematoxylin and eosin-stained, TUNEL-stained and calreticulin (CRT)-stained tumor tissues on day 14.

Article Snippet: After washing, the membranes were incubated with CD47 Polyclonal antibody (Proteintech Group, 20305-1-AP), PLK1 Polyclonal antibody (Proteintech Group, 10305-1-AP) or GAPDH Monoclonal antibody (Proteintech Group, 60004-1-Ig) for 1 h at room temperature.

Techniques: Expressing, Staining, TUNEL Assay