Journal: Acta Pharmaceutica Sinica. B

Article Title: Agonist-specific FPR1 conformational change prevents receptor recycling and promotes targeted protein degradation

doi: 10.1016/j.apsb.2026.03.019

Figure Lengend Snippet: Effect of fMLFC on FPR1 recycling. (A)Structure of fMLF and fMLFC. (B) HeLa FPR1–GFP, (C) RBL FPR1 stable cell line, (D) dHL-60 (differentiated HL-60) cells were treated with 5 μmol/L fMLF or fMLFC for 1 h at 37 °C to induce FPR1 internalization. In the FPR1 recycling group (fMLF REC and fMLFC REC), the cells were washed with PBS and resuspended in complete medium and incubated for 1 h at 37 °C for FPR1 recycling. The cell surface FPR1 was detected with the FPR1 antibody in the APC channel by flow cytometry. Data are presented as mean ± SEM ( n = 4 independent experiments). ∗∗ P < 0.01. (E) HeLa FPR1–GFP cells were treated with 5 μmol/L fMLF or fMLFC for 1 h at 37 °C to induce FPR1 internalization. In the FPR1 recycling group (fMLF REC and fMLFC REC), the cells were washed with PBS and resuspended in complete medium and incubated for 1 h at 37 °C for FPR1 recycling. Nuclei were stained with 2 μg/mL Hoechst (blue), and FPR1 was tagged with GFP (green). The experiments were performed on the confocal microscope with a 40 × oil objective. Scale bar = 25 μm.

Article Snippet: For FPR1 degradation assay, HeLa FPR1-GFP cells were pre-incubated for 1 h and kept in the presence of 1 μmol/L cycloheximide (CHX, YEASEN, Shanghai, China) then the cells were treated with lysosomal inhibitor chloroquine (CQ, 30 μmol/L), MG 132 (100 nmol/L) and bafilomycin A1 (BAF, 100 nmol/L) for 9 h at 37 °C in the presence of 5 μmol/L fMLFC.

Techniques: Stable Transfection, Incubation, Flow Cytometry, Staining, Microscopy

Journal: Acta Pharmaceutica Sinica. B

Article Title: Agonist-specific FPR1 conformational change prevents receptor recycling and promotes targeted protein degradation

doi: 10.1016/j.apsb.2026.03.019

Figure Lengend Snippet: Effect of SNX3 and SNX17 on FPR1 recycling. (A) HeLa cells were transfected with siRNA (siNC as a negative control, si SNX3 and si SNX17 ) for 48 h at 37 °C to knockdown the SNX expression before transfection with FPR1–GFP. The mRNAs of both si SNX3 and si SNX17 were significantly reduced. (B) Cell surface FPR1 expression with control (siNC) and specific siRNA knockdown, after agonist (5 μmol/L fMLF or fMLFC) stimulation for 1 h and recovery for another hour. Cell surface FPR1 expression was significantly reduced in cells receiving si SNX17 as detected by flow cytometry. (C) Confocal microscopic images showing the effect of siRNA knockdown on FPR1 recycling (1 h), after stimulation with 5 μmol/L fMLF or fMLFC for 1 h at 37 °C to induce FPR1 internalization. Nuclei were stained with Hoechst (blue), and FPR1 was tagged with GFP (green). Scale bar = 25 μm. (D, E) Colocalization of FPR1 and SNX3 (D) or FPR1 and SNX17 (E) in confocal microscopic images. The cells expressing FPR1 mRuby2 and Clover SNX protein were treated with 5 μmol/L fMLF or fMLFC for 1 h at 37 °C to induce FPR1 internalization, and then allowed for FPR1 recycling for 1 h. The green channel was SNX protein (Clover) and the red channel was FPR1 (mRuby2). Colocalization is indicated by yellow (overlay of red and green). Scale bar = 25 μm and 3 μm. (F) Fluorescence colocalization analysis. Pearson’s correlation coefficient ( R r) for colocalization of FPR1 mRuby2 and Clover SNX protein was calculated with Image J software plugin (Colocalization Finder). (G) Schematic diagram of the BRET assay using Nanoluc-tagged FPR1 and HaloTag–SNX17 (Promega). (H) HEK293T cells transfected to express FPR1–Nanoluc and HaloTag–SNX17 were incubated with the HaloTag ligand 618 (the 618 ligand) and then stimulated with either fMLF or fMLFC (5 μmol/L fMLF or fMLFC for 1h). The NanoLuc substrate furimazine was then added, and the BRET ratio was recorded at 460 and 615 nm. Data are presented as mean ± SEM ( n = 4 independent experiments). ∗∗ P < 0.01.

Article Snippet: For FPR1 degradation assay, HeLa FPR1-GFP cells were pre-incubated for 1 h and kept in the presence of 1 μmol/L cycloheximide (CHX, YEASEN, Shanghai, China) then the cells were treated with lysosomal inhibitor chloroquine (CQ, 30 μmol/L), MG 132 (100 nmol/L) and bafilomycin A1 (BAF, 100 nmol/L) for 9 h at 37 °C in the presence of 5 μmol/L fMLFC.

Techniques: Transfection, Negative Control, Knockdown, Expressing, Control, Flow Cytometry, Staining, Fluorescence, Software, Bioluminescence Resonance Energy Transfer, Incubation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Agonist-specific FPR1 conformational change prevents receptor recycling and promotes targeted protein degradation

doi: 10.1016/j.apsb.2026.03.019

Figure Lengend Snippet: FPR1 degradation pathways. (A) fMLFC induced FPR1 degradation. HeLa cells expressing FPR1–GFP were pre-incubated with 1 μmol/L cycloheximide for 1 h, and then were added 5 μmol/L fMLF or fMLFC with or without 30 μmol/L lysosomal inhibitor chloroquine (CQ) for another 9 h of incubation at 37 °C. Then, the remaining FPR1–GFP was detected based on green fluorescence by flow cytometry. (B–D), colocalization of FPR1 with the early endosome marker RAB5 (B), the late endosome marker RAB7. (C) And the lysosome marker LAMP1 (D), These markers were fluorescent-tagged fusion proteins as marked and co-transfected with FPR1 for 24 h at 37 °C. The experiments were performed on a confocal microscope. Scale bar = 25 and 3 μm. (E) Fluorescence colocalization analysis. Pearson’s correlation coefficient ( R r) for colocalization of FPR1 and RAB5, RAB7 or LAMP1 were calculated with Image J software plugin (Colocalization Finder). Data are presented as mean ± SEM ( n = 4 independent experiments). ∗∗ P < 0.01.

Article Snippet: For FPR1 degradation assay, HeLa FPR1-GFP cells were pre-incubated for 1 h and kept in the presence of 1 μmol/L cycloheximide (CHX, YEASEN, Shanghai, China) then the cells were treated with lysosomal inhibitor chloroquine (CQ, 30 μmol/L), MG 132 (100 nmol/L) and bafilomycin A1 (BAF, 100 nmol/L) for 9 h at 37 °C in the presence of 5 μmol/L fMLFC.

Techniques: Expressing, Incubation, Fluorescence, Flow Cytometry, Marker, Transfection, Microscopy, Software

Journal: The Journal of Clinical Investigation

Article Title: PARP inhibitors restore NK cell function via secretory crosstalk with tumor cells in prostate cancer

doi: 10.1172/JCI197157

Figure Lengend Snippet: ( A ) Tumor tissues were collected 14 days after inoculation with RM-1 cells in mice. TILs and TINKs were isolated. TILs were cultured with recombinant proteins in vitro, and recombinant protein tags were detected via flow cytometry. TINKs were cultured with recombinant proteins in vitro, and binding proteins were identified by IP-MS. FC, flow cytometry. ( B ) Venn diagram showing proteins with IP-MS scores ≥ 100 in TINKs, PBNKs, and PCa cells. ( C ) Proportion of membrane proteins versus nonmembrane proteins among overlapping proteins from B . ( D ) Membrane protein profiles identified by IP-MS. ( E ) Co-IP demonstrating mutual binding between ANXA6 and CypA-His. IB, immunoblotting. ( F ) Protein docking prediction between ANXA6 and CypA proteins using AlphaFold3. ( G ) Western blot analysis of ANXA6 expression and phosphorylation levels in NK cells treated with CypA and/or CsA. ( H ) Co-IP analysis of ANXA6 and FPR1 interaction in CypA-treated NK cells. ( I ) Fpr1 fl/fl mice were crossed with the Ncr1-iCre transgenic mice to generate the NK cell–specific Fpr1 -KO mice, which are denoted as Ncr1-iCre + -Fpr1 fl/fl . ( J ) Tumor growth curves and tumor weights in Fpr1 fl/fl and Ncr1-iCre + -Fpr1 fl/fl (cKO) mice inoculated with RM-1 vehicle or CypA-OE cells, respectively; N = 5 per group. Tumor growth curve data are presented as mean ± SD and were analyzed by 2-way ANOVA with Tukey’s multiple-comparison test. Tumor weight data are presented as mean ± SEM and were analyzed by 1-way ANOVA.

Article Snippet: 6- to 8-week-old male WT C57BL/6J mice, FVB mice, and NOG mice were purchased from Charles River Co., Ltd. Ncr1-iCre mice and Fpr1 fl/fl mice were purchased from Cyagen Biosciences.

Techniques: Isolation, Cell Culture, Recombinant, In Vitro, Flow Cytometry, Binding Assay, Protein-Protein interactions, Membrane, Co-Immunoprecipitation Assay, Western Blot, Expressing, Phospho-proteomics, Transgenic Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: PARP inhibitors restore NK cell function via secretory crosstalk with tumor cells in prostate cancer

doi: 10.1172/JCI197157

Figure Lengend Snippet: ( A ) Tumor growth curves and weights in PARPi-treated tumor-bearing mice coadministered with CypA inhibitor CsA and FPR1 inhibitors CsH and HCH6-1; N = 6 per group; scale bars: 1 cm ( B ) Tumor growth curves and weights in C57BL/6 mice bearing RM-1 tumors treated with FPR1 agonist fMIFL or inhibitor HCH6-1; N = 5 per group; scale bars: 1 cm ( C and D ) IFN-γ and GZMB expression levels in TINKs ( C ) and their quantification ( D ) under HCH6-1 or fMIFL treatment; N = 5 per group. ( E ) Western blot analysis of ERK- and AKT-related pathway activation in NK cells treated with CypA, CsA, CsH, or HCH6-1 in vitro. ( F and G ) Flow cytometry analysis of IFN-γ and GZMB expression ( F ) and quantification ( G ) in NK cells treated with FPR1 inhibitor CsH, AKT inhibitor MK-2206, or ERK inhibitor SCH772984; N = 5 per group. ( H ) Transmission electron microscopy images showing mitochondrial structure and crista alterations in NK cells stimulated with CypA. Scale bars: 1 μm. ( I ) Statistical analysis of mitochondrial number ( N = 10) and crista count ( N = 15) in NK cells. Tumor growth curve data are presented as mean ± SD and were analyzed by 2-way ANOVA with Tukey’s multiple-comparison test. Other data are presented as mean ± SEM and were analyzed by 1-way ANOVA ( A , B , D , and G ) and Welch’s t test ( I ).

Article Snippet: 6- to 8-week-old male WT C57BL/6J mice, FVB mice, and NOG mice were purchased from Charles River Co., Ltd. Ncr1-iCre mice and Fpr1 fl/fl mice were purchased from Cyagen Biosciences.

Techniques: Expressing, Western Blot, Activation Assay, In Vitro, Flow Cytometry, Transmission Assay, Electron Microscopy, Comparison