Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 1. CCR2 is required for efficient SFTSV infection in cells. (A to C) RT-qPCR (A), flow cytometry (B), and Western blot (C) analysis of SFTSV infection at 24 hours after infection in CCR2- or ATF6-knockdown THP-1 cells. n = 6. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) RT-qPCR analysis of virus RNA in CCR2-KO THP-1 cells inoculated with four phylogenetically distinct SFTSV strains, including HBMC16, HNXY2017-50, HNXY2017-66, and WCH, for 24 hours. n = 4. (E) Multistep growth curves of four SFTSV strains in CCR2-KO THP-1 cells. n = 4. (F) Surface expression of CCR2 on BMDMs from CCR2−/−and WT C57BL/6J mice. A representative of three replicates is shown. (G and H) Statistical results (G) and scanned images (H) of the immunological focus assay of SFTSV titers at 24 hours after infection in BMDMs with deletions in CCR2. n = 6. (I) Microscopy of BMDMs immunostained for F4/80, SFTSV NP, and DAPI at 24 hours after infection. (J) SFTSV infection rates at 24 hours after infection determined by flow cytometry analysis in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. (K) Representative flow plot of SFTSV infection in Huh7 cells. (L) Supernatant viral titers measured by immunological focus assay in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (G)]. One-way ANOVA followed by Tukey’s multiple comparisons test was per- formed for comparison of variables among three groups [(J) and (K)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Quantitative RT-PCR, Flow Cytometry, Western Blot, Knockdown, Virus, Expressing, Microscopy, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 2. Effect of CCR2 inhibitor and antibody on SFTSV infection in cells. (A to F) Effects of CCR2 antagonist RS102895 and CCR2 antagonist 1 on SFTSV infection in THP-1 (A) to (C) and Huh7 cells (D) to (F). At 24 hours after infection, relative vRNA levels were measured via RT-qPCR [(A), (B), (D), and (E), n = 3], and relative intracellular SFTSV NP levels were measured by Western blotting (C) and (F). Cell viability was measured using CCK-8 [(D) and (E), n = 3]. (G to I) Effects of CCR2 antibody on SFTSV infection in THP-1 cells. The dose-dependent inhibitory effects of the CCR2 antibody were analyzed by detecting virus loads in THP-1 cells at 24 hours after infection [(G), n = 6]. SFTSV infection rates were measured at 24 hours after infection with MOIs of 1 and 5 [(H), n = 6]. Representative flow plot of SFTSV infection in THP-1 cells treated with anti-human CCR2 or isotype control antibody (I). Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), (E), (G), and (H)]. R2 [(A), (B), (D), and (E)] was estimated by a nonlinear regression model (curve fit).

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Virus, Control, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 3. CCR2 mediates SFTSV binding and internalization. (A and B) Effects of CCR2 deletions on the internalization (A) and binding (B) of SFTSV in BMDMs. Relative vRNA levels were measured via RT-qPCR. n = 6. (C) Fluorescence microscopy analysis of the effects of CCR2 deletions on the binding of SFTSV. BMDMs were immunos- tained with F4/80 (green), SFTSV NP (red), and DAPI. A representative of three replicates is shown. (D and E) SFTSV binding assay in CCR2-KO (KO) THP-1 cells by using RT- qPCR (D) and immunofluorescence staining (E). n = 4. Cellular membranes were labeled with WGA. (F) Flow cytometry analysis of SFTSV infection at 2 hours after infection in Huh7, HeLa, and Jurkat cells overexpressing CCR2A or CCR2B. n = 6. (G to I) Effects of the CCR2 antagonist RS102895 (G), CCR2 antagonist 1 (H), and favipiravir (I) on the binding and internalization of SFTSV in THP-1 cells. n = 3. (J) Binding assay of HRTV, RVFV, and AMRV for control and CCR2-KO THP-1 cells. n = 3. (K and L) Effects of CCR2 antagonist RS102895 (K) and CCR2 antagonist 1 (L) on the binding of HRTV, RVFV, and AMRV in THP-1 cells. n = 3. Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (J)]. One-way ANOVA followed by Tukey’s multiple comparisons test was performed for comparison of variables among three groups [(F) to (I), (K), and (L)]. ns, no significance; p.i., post-infection.

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Binding Assay, Quantitative RT-PCR, Fluorescence, Microscopy, Immunofluorescence, Staining, Labeling, Flow Cytometry, Infection, Control, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 4. The CCR2 N-terminal extracellular domain mediates SFTSV binding to cells. (A) Coimmunoprecipitation of co-overex- pressed Gn-strep protein and CCR2-flag proteins in HEK293T cells using strep-tag binding beads or anti-Flag antibody beads. Vector-flag, GFP-flag, and SCARB1-flag pro- teins were used as negative controls. (B to D) Effect of CCR2 overexpression on the in- fectivity of SFTSV in Huh7 (B), HeLa (C), and Jurkat (D) cells. SFTSV infection rates were measured at 24 hours after infection by flow cytometry analysis in control-, CCR2A-, CCR2B-, CCR2A-ΔN–, and CCR2B-ΔN–over- expressing cells. N = 6. (E) Surface expres- sion of CCR2 and representative flow plot of SFTSV infection in Jurkat cells. (F and G) Effect of the CCR2 N-terminal extracellular domain on the infectivity of SFTSV in HeLa (F) and Jurkat (G) cells. SFTSV infection rates were measured at 24 hours after infection by flow cytometry in control-, CCR2A-, CCR2A-N14Q–, CCR2A-Y26F–, CCR2B-, CCR2B-N14Q–, and CCR2B-Y26F–overex- pressing cells. n = 6. (H) Binding of CCR2 N- terminal–derived Y26 sulfated peptide (p2), peptide without tyrosine sulfation at Y26 (p1), or the scrambled peptide to SFTSV virions determined by PRM assay. Statistical analysis is shown in the left panel, and PRM transitions are shown in the right panel. (I and J) Effect of CCR2 N-terminal–derived peptides on the infectivity of SFTSV in THP-1 cells. Relative intracellular vRNA levels and supernatant viral titers were measured at 24 hours after infection via RT-qPCR. n = 4. (K) Effect of CCR2 N-terminal–derived peptides on the binding of SFTSV in THP-1 cells. Rel- ative levels of bound virions were measured via RT-qPCR. n = 4. One-way ANOVA fol- lowed by Tukey’s multiple comparisons test was performed for comparison of variables among three groups [(B) to (D) and (F) to (K)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Binding Assay, Strep-tag, Plasmid Preparation, Over Expression, Infection, Flow Cytometry, Control, Expressing, Derivative Assay, Quantitative RT-PCR, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 5. CCR2 contributes to SFTSV pathogenesis in mouse models. (A) Serum and spleen viral titers in SFTSV-infected CCR2−/−and WT C57BL/6J mice (four for each group) tested by immunological focus assay at 3 and 5 dpi. (B and C) Survival probability (B) and relative body weight (C) in anti-IFNAR1 antibody–pretreated CCR2−/−(n = 11) and WT (n = 11) C57BL/6J mice after intraperitoneal infection with SFTSV. (D) Viral titers in serum, spleen, liver, and lung samples from anti-IFNAR1 antibody–pre- treated CCR2−/−and WT C57BL/6J mice (four for each group) tested by immunological focus assay at 3 and 5 dpi. (E and F) Representative images of spleen, liver, and lung sections collected at 5 dpi from control and SFTSV-challenged CCR2−/−and WT C57BL/6J mice stained with a rabbit polyclonal antibody against SFTSV NP (E) or with hematoxylin and eosin (F). (G) Viral titers in serum from anti-IFNAR1 antibody–pretreated C57BL/6J mice tested by immunological focus assay at 3 (n = 13 for each group) and 5 (n = 10 for nontreated group and n = 13 for treated group) dpi. (H) Survival probability in anti-IFNAR1 antibody–pretreated C57BL/6J mice with SFTSV infection in the absence (n = 13) or presence (n = 13) of CCR2 antagonist RS102895 and without SFTSV infection (n = 5). Two-tailed Student’s t test was performed for comparison of variables between two groups [(A), (B), (D), and (G)]. The Kaplan-Meier method was used to analyze time-to-event data [(C) and (H)].

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Control, Staining, Two Tailed Test, Comparison

Journal: Science advances

Article Title: CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

doi: 10.1126/sciadv.adg6856

Figure Lengend Snippet: Fig. 6. CCR2 contributes to SFTSV infectivity in primary human monocytes. (A and B) Comparisons of surface CCR2 expression levels on primary human mono- cytes (left) and the ability of SFTS binding (right) between donors aged <60 (n = 10) and ≥60 (n = 10) years old (A), as well as between healthy donors (n = 8) and donors with DM (n = 8) (B). MFI, mean fluorescent intensity. (C) Association of surface CCR2 expression level on primary human monocytes with virus binding ability or with the age of donors (n = 20). Ra 2 indicates the correlation between the CCR2 expression level and individual age, and Rb 2 indicates the correlation between the CCR2 expression level and the binding ability of SFTSV. (D) Associa- tion of surface CCR2 expression level on primary human monocytes with the peak viral load in serum that was consecutively collected from SFTS patients (n = 45) during the clinical course. Two-tailed Student’s t test was performed for compar- ison of variables between two groups [(A) and (B)]. R2 [(C) and (D)] was estimated by a linear regression model. HC, healthy control; DM, diabetes mellitus; CT, cycle threshold.

Article Snippet: CCR2 antagonist and anti-human CCR2 antibody inhibition assays THP-1 and Huh7 cells were pretreated with serial concentrations of two CCR2 antagonists, CCR2 antagonist RS102895 hydrochloride (MedChemExpress, catalog no. 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, catalog no. 1683534-96-4), and an antihuman CCR2 antibody (BioLegend, catalog no. 357202) or IgG isotype control (BioLegend, catalog no. 400201).

Techniques: Infection, Expressing, Binding Assay, Virus, Two Tailed Test, Control

Journal: Cell reports

Article Title: Inflammation induces pro-NETotic neutrophils via TNFR2 signaling.

doi: 10.1016/j.celrep.2022.110710

Figure Lengend Snippet: Figure 5. Increased ELANE expression in CCR5+ pPMNs (A and B) The expression of ELANE was quantified by intracellular fluorescent immunolabeling (A). A statistical evaluation is shown in (B) (n = 3; SEM; ANOVA; *p < 0.05). (C–E) The subcellular localization of ELANE was analyzed using imaging flow cytometry (C). Nuclear localization of ELANE was quantified by calculation of the similarity score provided by imaging flow cytometry (D), and statistics were calculated for three independent experiments (E; n = 3; SEM; t test; *p < 0.05).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CCR5-PE Miltenyi Cat# 130-117-356; RRID: AB_2733783 CCR5 unlabeled R&D Cat# MAB182;RRID:AB_2072686 CCR4-BV605 Biolegend 359418; RRID:AB_2562483) CCR1-APC-Fire 750 Biolegend Cat# 362915; RRID:AB_2734402 CXCR1-APC Biolegend Cat# 320612; RRID:AB_2126475 CD66b-PerCP-Cy5.5 Biolegend Cat# 305108; RRID:AB_2631503 CD15-AF594 Biolegend Cat# 301917; RRID:AB_2632827 CD62L-FITC Biolegend Cat# 304803;RRID:AB_314463 CD49d-APC Biolegend Cat# 304308; RRID:AB_2130041 CD16-APC-Cy7 Biolegend Cat# 302018; RRID:AB_314218 HLA-DR-AF700 Biolegend Cat# 307625; RRID:AB_493770 TNFa-PE-Dazzle594 Biolegend Cat# 502946; RRID:AB_2564173 TNFR1-APC Biolegend Cat# 369906; RRID:AB_2650764 TNFR2-PE-Cy7 Biolegend Cat# 358412; RRID:AB_2564396 CXCR3-APC BD Bioscience Cat# 550967; RRID:AB_398481 CXCR3-PE-Cy5 BD Bioscience Cat# 551128; RRID:AB_394061 CXCR4-PE-Cy5 BD Bioscience Cat# 555975; RRID:AB_396268 CCR3-PE BD Bioscience Cat# 558165; RRID:AB_10611864 CCR3-PE-CF594 BD Bioscience Cat# 562571; RRID:AB_2737660 CD15-BV605 BD Bioscience Cat# 562980; RRID:AB_2744292 CD49d-PE-Cy5 BD Bioscience Cat# 559880; RRID:AB_397355 MPO-PE BD Bioscience Cat# 333139; RRID:AB_2868640 CXCR4-FITC R&D Systems Cat# FAB170F;RRID:AB_357075 Azurocidin Abcam Cat# ab167336; RRID:AB_2040581 Anti-mouse-Cy5 Dianova Cat# 715-175-150; RRID:AB_2340819 Histone H4 (citrulline 3) EMD Millipore Cat# 07-596; RRID:AB_441947 TNF neutralizing Antibody Sino Biologicals Cat# 10602-R10N1;RRID:AB_2860205) Bacterial and virus strains Ampicillin-resistant, GFP-expressing Escherichia coli E. coli: One ShotTM TOP10 Chemically Competent E. coli GFP-vector: pAcGFP1 ThermoFisher Takarabio C404010 632468 Biological samples Healthy donor PMN UniversityHospital Heidelberg NA Colonic tissue samples UniversityHospital Heidelberg NA Peripheral blood of IBD patients UniversityHospital Heidelberg NA Chemicals, peptides, and recombinant proteins DAPI Merck Sigma-Aldrich D9542 Phalloidin Thermo Fisher Scientific A12379 Ionomycin Merck Sigma-Aldrich IO634 Paraformaldehyde Merck Sigma-Aldrich 818715 Cytochalasin D Merck Sigma-Aldrich C8273 PMA Merck Sigma-Aldrich P8139 (Continued on next page) e1 Cell Reports 39, 110710, April 19, 2022

Techniques: Expressing, Immunolabeling, Imaging, Cytometry

Journal: Discover Oncology

Article Title: Integrated WGCNA retrieval of T-cell exhaustion genes related to radiosensitivity in locally advanced cervical cancer and prediction of immunotherapy efficacy

doi: 10.1007/s12672-025-03673-y

Figure Lengend Snippet: IHC assay results of the protein expression levels of APIBEC3H, CCR7, PILRA, and SLC15A3 in LACC patients

Article Snippet: Subsequently, the sections were incubated at 4 °C overnight with the corresponding primary antibody against APOBEC3H (1:200, PA5-54426, Invitrogen, USA), CCR7 (1:100, 25898-1-AP, Proteintech, China), PLRA (1:100, PA5-147474, Invitrogen, USA), SLC15A3 (1:100, PA5-61614, Invitrogen, USA).

Techniques: Expressing

Journal: Experimental Biology and Medicine

Article Title: Engineering ADSCs by manipulating YAP for lymphedema treatment in a mouse tail model

doi: 10.3389/ebm.2024.10295

Figure Lengend Snippet: VEGFC successfully induced lymphatic endothelial transdifferentiation of ADSCs. (A–C) PCR tests showed the upregulation of VEGFR-3, Prox-1, Lyve-1 during the lymphatic endothelial transdifferentiation of ADSCs. (D) VEGFR-3 as a typical LEC marker was detected by immunofluorescence staining after VEGFC-induction at the indicated times. Scale bar = 50 μm. (E) Immunofluorescence intensity analysis of VEGFR-3. (F, H) Western blot showed the increased VEGFR-3 expression during the lymphatic endothelial transdifferentiation of ADSCs. (G) ADSCs were seeded on Matrigel after 7-day induction, and tube formation was evaluated at 12 h postseeding. VEGFC group generated tube-like structure while control group did not exhibit tubes. Scale bar = 100 μm. Bars: means ± standard deviation. n = 3 in each group, ns: no significant, ** P < 0.01, *** P < 0.001.

Article Snippet: For the lymphatic transdifferentiation of ADSCs, VEGFC (100 ng/mL, HY-P77864, MedChem Express, Monmouth Junction, NJ, United States) was used for 7 days.

Techniques: Marker, Immunofluorescence, Staining, Western Blot, Expressing, Generated, Control, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: Engineering ADSCs by manipulating YAP for lymphedema treatment in a mouse tail model

doi: 10.3389/ebm.2024.10295

Figure Lengend Snippet: Effect of lymphatic endothelial transdifferentiation and verteporfin on the expression of YAP. (A) Immunostaining of YAP in the control group and VEGFC group. Scale bar = 50 μm. (B) Immunofluorescence intensity analysis of YAP. (C–E) Western blot showed the decreased nuclear YAP expression and increased cytosolic YAP expression in ADSCs after lymphatic endothelial transdifferentiation. (F) PCR test showed that verteporfin suppressed the expression of YAP in ADSCs at the concentration of 20 μM. (G , H) Western blot showed the continuously inhibitory effect of verteporfin on YAP expression in ADSCs. Bars: means ± standard deviation. n = 3 in each group; ns, no significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For the lymphatic transdifferentiation of ADSCs, VEGFC (100 ng/mL, HY-P77864, MedChem Express, Monmouth Junction, NJ, United States) was used for 7 days.

Techniques: Expressing, Immunostaining, Control, Immunofluorescence, Western Blot, Concentration Assay, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: Engineering ADSCs by manipulating YAP for lymphedema treatment in a mouse tail model

doi: 10.3389/ebm.2024.10295

Figure Lengend Snippet: The downregulation of YAP enhanced the lymphatic endothelial transdifferentiation of ADSCs in vitro . 20 μM verteporfin preconditioning for 48 h downregulated the expression of YAP in ADSCs. Under this inhibitory effect, higher expression levels of VEGFR-3 can be detected after differentiation of ADSCs, and larger density of tube formation can be observed. (A) Lymphatic endothelial transdifferentiation of ADSCs was conducted after 20 μM-verteporfin preconditioning for 48 h, and immunofluorescence staining indicated higher level of VEGFR-3. Scale bar = 50 μm. (B) Immunofluorescence intensity analysis of VEGFR-3. (C, D) Higher expression level of VEGFR-3 was confirmed by western blot. (E, F) The tube formation assay showed that VEGFC (+) verteporfin (+) group generated more tube-like structure than VEGFC (+) verteporfin (−) group. And quantification of master segments was analysed. Scale bar = 200 μm. Bars: means ± standard deviation. n = 3 in each group, ns: no significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For the lymphatic transdifferentiation of ADSCs, VEGFC (100 ng/mL, HY-P77864, MedChem Express, Monmouth Junction, NJ, United States) was used for 7 days.

Techniques: In Vitro, Expressing, Immunofluorescence, Staining, Western Blot, Tube Formation Assay, Generated, Standard Deviation

Journal: Cell chemical biology

Article Title: Degradome analysis to identify direct protein substrates of small-molecule degraders.

doi: 10.1016/j.chembiol.2024.10.007

Figure Lengend Snippet: Figure 3. Proteome response to CC-885 treatment (A) Label-free whole proteome, (B) degradome, and (C) nascent proteome analysis for GSPT1 degrader CC-885. Jurkat cells were treated with 100 nM CC- 885 for 8 h in all three approaches. Volcano plots show log2 protein fold change vs. nominal p values. Highlighted in yellow are three known targets of CC-885, and in red all proteins that were down- regulated in the whole proteome dataset (log2FC (treated/untreated) < 1 and log10(p-value) > 1). See also Figure S2.

Article Snippet: MLN4924 (THP Medical #HY-70062), Carfilzomib (Selleck Chemicals #S2852), TAK243 (MedChem Express #HY-100487), CC220 (Selleck Chemicals #S8760), CC122 (Selleck Chemicals #S7892), Thalidomide (Sigma-Aldrich #T144), Lenalidomide (SigmaAldirch #SML2283), Pomalidomide (THP Medical Product #HY-10984), CC-885 (MedChem Express #HY-101488), CC-90009 (MedKoo #207005).

Techniques:

Journal: Cell chemical biology

Article Title: Degradome analysis to identify direct protein substrates of small-molecule degraders.

doi: 10.1016/j.chembiol.2024.10.007

Figure Lengend Snippet: Figure 4. Validation of FIZ1 as a degradation target of compound 1 (A) Chemical structure of small molecule degrader compound 1. (B) Volcano plot of degradome proteomics under compound 1 treatment (log2 protein fold change vs. nominal p values). Jurkat cells were treated for 13 h with 5 mM compound 1 or DMSO. Downregulated proteins are highlighted in yellow. (C) Degradation of IKZF3-nluc-FLAG and endogenous IKZF3 in response to compound 1 treatment determined by immunoblot analysis. Jurkat cells were treated with 10 mM compound 1 or DMSO for 15 h. Antibodies used: anti-FLAG, anti-IKZF3, and anti-vinculin (loading control). (D and E) Degradation of FIZ1-nluc-FLAG in response to compound 1 treatment determined by immunoblot analysis in (D) Hek293T cells and (E) in Jurkat cells. Cells were treated with 10 mM compound 1 or DMSO for 16 h. Antibodies used: anti-FLAG, anti-IKZF3 (positive control), and anti-vinculin (loading control). (F) Luminescence assay in HEK293T cells treated with either compound 1 (5 mM, 20 mM) or DMSO for the indicated times. Error bars represent standard errors of the mean. (G) Degradation of FIZ1-nluc-FLAG in response to 20 mM compound 1 treatment for 13 h in HEK293T cells under inhibition of neddylation (1 mM MLN4924 = NAE inhibitor), the proteasome (1 mM carfilzomib) or ubiquitination (10 mM TAK243 = UBA1 inhibitor), determined by immunoblot analysis. Upper panel: anti-FLAG antibody. Lower panel: anti-vinculin (loading control). (H) Degradation of FIZ1-nluc-FLAG in the presence and absence of CRBN. Monoclonal CRBN knockout RKO cells with and without reconstituted wild-type CRBN and transduced with a lentiviral plasmid for stable expression of FIZ1-NanoLuc-3xFLAG were treated with DMSO or 10 mM of compound 1, CC-90009, or pomalidomide for 24 h. (I) Degradation of FIZ1-nluc-FLAG in response to different small molecule degraders determined by immunoblot analysis. HEK293T cells were treated with DMSO, 20 mM compound 1, 10 mM CC220, 10 mM CC122, 10 mM thalidomide, 10 mM lenalidomide, 10 mM pomalidomide, 10 mM CC-885, or 10 mM CC-90009 for 15 h.

Article Snippet: MLN4924 (THP Medical #HY-70062), Carfilzomib (Selleck Chemicals #S2852), TAK243 (MedChem Express #HY-100487), CC220 (Selleck Chemicals #S8760), CC122 (Selleck Chemicals #S7892), Thalidomide (Sigma-Aldrich #T144), Lenalidomide (SigmaAldirch #SML2283), Pomalidomide (THP Medical Product #HY-10984), CC-885 (MedChem Express #HY-101488), CC-90009 (MedKoo #207005).

Techniques: Biomarker Discovery, Western Blot, Control, Positive Control, Luminescence Assay, Inhibition, Ubiquitin Proteomics, Knock-Out, Transduction, Plasmid Preparation, Expressing

Journal: Journal for immunotherapy of cancer

Article Title: Propafenone facilitates mitochondrial-associated ferroptosis and synergizes with immunotherapy in melanoma.

doi: 10.1136/jitc-2024-009805

Figure Lengend Snippet: Figure 5 JNK/JUN signaling-mediated HMOX1 upregulation promotes mitochondria-associated ferroptosis. (A) Relative mRNA levels of HMOX1 in A375 and SK-MEL-28 cells treated with RSL3 alone or in combination with PPF, compared with the DMSO group, were assessed in the presence of CHX (5 µg/mL). (B) Venn diagram showing the putative upstream transcription factors of HMOX1 predicted by JASPAR, PROMO, and hTFtarget databases. (C) Correlation between JUN and HMOX1 using Spearman’s correlation in cell lines from the CCLE database. (D) Correlation between JUN and WP_ferroptosis score using Spearman’s correlation in cell lines from the CCLE database. (E) JUN mRNA level in A375 and SK-MEL-28 cells after indicated treatment. (F) A375 and SK-MEL-28 cells were treated with PPF, RSL3 or a combination for 3 hours and subjected to immunofluorescence microscopy with antibodies against JUN. Nuclei counterstained by DAPI (blue). Scale bar, 50 µm. (G) GSEA shows the enrichment of the JNK cascade pathway (blue) and MAPK pathway (red) between the co-treatment group and the RSL3 group in RNA sequencing. (H–I) The relative viability of A375 and SK-MEL-28 cells treated with the combination of PPF and RSL3 with or without compounds from the kinase drug library for 6 hours. Specific compounds are annotated. (J) Cell viability of A375 and SK-MEL-28 cells at different time points after co-treatment of PPF and RSL3 in the absence or presence of tanzisertib (5 µM, 10 µM or 15 µM). (K–M) Cell death of A375 and SK-MEL-28 cells induced by the indicated treatment for 6 hours was shown using fluorescence microscopy and quantified by PI-staining coupled with flow cytometry. Representative images are presented at 200× magnification. PPF, 5 µM; RSL3, 2.5 µM; tanzisertib, 10 µM. (N) Mitochondrial ferrous iron levels were assessed by flow cytometry in A375 and SK-MEL-28 cells after the indicated treatment for 1 hour. (O) Genome browser view of binding peaks on the HMOX1 promoter in JUN ChIP and input control of MDA-MB-231 cells and MDA-MB-231–1833 cells based on data from GSE112444. P values were calculated using one-way analysis of variance analysis. ***p<0.001. CCK- 8, Cell Counting Kit-8; CCLE, Cancer Cell Line Encyclopedia; ChIP, chromatin immunoprecipitation; CHX, cycloheximide; COM, combination; DMSO, dimethyl sulfoxide; GSEA, Gene Set Enrichment Analysis; HOMX1, heme oxygenase 1; JUN/JNK, Jun N- terminal kinase (JNK)/JUN; MDA, malondialdehyde; mRNA, messenger RNA; NES, normalized enrichment score; PI, propidium iodide; PPF, propafenone; TPM, transcripts per million.

Article Snippet: D ow nloaded from 15Zhou Q, et al. J Immunother Cancer 2024;12:e009805. doi:10.1136/jitc-2024-009805 Open access Z- VAD- FMK (HY- 16658B), DFO (HY- B1625), Fer- 1 (HY100579), TEMPO (HY- W001187), Mito- TEMPO (HY112879), hemin (HY- 19424), ZnPP (HY- 101193), CHX (HY- 12320), tanzisertib (HY- 15495), trigonelline (HYN0414) and ML385 (HY- 100523) were purchased from MedChemExpress.

Techniques: Immunofluorescence, Microscopy, RNA Sequencing, Drug discovery, Fluorescence, Staining, Flow Cytometry, Binding Assay, Control, CCK-8 Assay, Cell Counting, Chromatin Immunoprecipitation