Journal: Autophagy

Article Title: Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

doi: 10.1080/15548627.2025.2477385

Figure Lengend Snippet: MCPyV truncated LT induces paranuclear retention and stabilization of KIT. (A) Immunofluorescence detection of KIT (green) and MCPyV LT (CM2B4, red) or sT (CM8E6, red) in KIT-HEK293 cells transfected with MCPyV T-antigens or vector control ( n = 6). (B) Top : illustration of the expression constructs of LT339 and the VPS39-interaction defective mutant LT339 W209A . Bottom : Representative images showing the effect of LT339 and LT339 W209A mutant on localization of KIT (green). LT was detected by CM2B4 (red). ( n = 3) (C) Immunoblots showing the effect of the LT339 and LT339 W209A on KIT expression. The quantification of KIT level is shown below the immunoblots ( n = 9). (D) Top : immunoblot analysis of the effect LT339 and LT339 W209A on KIT protein stability in the presence of cycloheximide (CHX) up to 4 h. Bottom : quantification of KIT protein stability after normalization to 0 h time point ( n = 4). (E) Immunoblots showing KIT protein stability in KIT-HEK293 cells transfected with LT339, LT339 W209A or plasmid control (CTR) treated with KIT ligand (KITLG, 100 ng/mL) or solvent control (PBS) in the presence of CHX (100 μg/mL). (F) Quantification of KIT protein stability after normalization to 0 h time point ( n = 5). Solid lines represent PBS control (KITLG − ) and dotted lines represent KITLG treatment (KITLG + ). (A and B) Nuclei were stained by DAPI (blue). Scale bar: 10 μm. Numbers below the images refer to the proportion of cells with KIT paranuclear dot-like staining to the total number of cells analyzed. (C, D and F) Error bars represent mean ± SEM. * p < 0.05, *** p < 0.001, ns = not significant were calculated by one-way ANOVA with post-hoc Tukey’s test (C), two-way ANOVA (D) or two-way ANOVA with post-hoc Bonferroni’s test (F).

Article Snippet: For KITLG induced KIT degradation experiments, KIT-293 cells were transfected with LT339, LT339 W209A or CTR for 48 h, followed by addition of CHX (100 μg/mL) and KITLG (100 ng/mL; Proteintech Group, HZ-1024) or PBS.

Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Control, Expressing, Construct, Mutagenesis, Western Blot, Solvent, Staining

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Brief methods of bone marrow-derived macrophage induction.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Transcriptomic comparison of porcine macrophage phenotypes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) versus M2 (M1_IFNγ + LPS, and M1_GM-CSF). ( A ) PCA clustering M1_IFNγ + LPS ( n = 4), M1_GM-CSF ( n = 4), M2_IL4 + IL10 ( n = 3), M2_M-CSF ( n = 3); ( B ) Top enriched GO biological processes of 730 DEG genes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) and M2 (M2_IL4 + IL10, and M2_M-CSF); ( C ) Heatmap exhibiting 49 classical macrophage marker genes expression profile among M1_IFNγ + LPS, M1_GM-CSF, M2_IL4 + IL10, and M2_M-CSF; ( D ) The DEGs between M1 and M2 were studied by using the String online tool. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Comparison, Marker, Expressing

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Comparison of transcriptomics in different phenotypes of porcine macrophages. ( A ) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method, M1_IFNγ + LPS, and M1_GM-CSF; ( B ) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method M2_IL4 + IL10 and M2_M-CSF; ( C ) GO pathway enrichment analysis of two polarized macrophages, M1_IFNγ + LPS and M1_GM-CSF; ( D ) GO pathway enrichment analysis of two polarized macrophages, M2_IL4 + IL10 and M2_M-CSF; ( E ) The DEGs were studied by using the String online tool, M1_IFNγ + LPS, and M1_GM-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 1; ( F ) The DEGs are studied by using the String online tool, M2_IL4 + IL10, and M2_M-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 2.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Comparison

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Validation of M1_IFNγ + LPS, M1_GMCSF, M2_M-CSF, and M2_IL4 + IL10 signatures. ( A ) SS2-infected macrophages were enriched in M1_IFNγ + LPS based on GSEA (Data source: Microarray analysis); ( B ) PRRSV-infected macrophages were enriched in M1_GM-CSF based on GSEA (Data source: RNA-seq); ( C ). T. gondii -infected macrophages were enriched in M2_IL4 + IL10 based on GSEA (Data source: RNA-seq); ( D ) T. gondii Me49 -infected macrophages were enriched in M2_M-CSF based on GSEA (Data source: RNA-seq).

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Biomarker Discovery, Infection, Microarray, RNA Sequencing

Journal: BMC Pulmonary Medicine

Article Title: β-elemene inhibits tumor-promoting in small cell lung cancer by affecting M2 macrophages and TGF-β

doi: 10.1186/s12890-025-03533-z

Figure Lengend Snippet: The Effect of M2 Macrophages on NCI-H209 Cells. ( A ) PMA-induced differentiation of THP-1 into M0 macrophages. ( B and C ) IL-4 and IL-13 induce the differentiation of M0 macrophages into M2 macrophages. ( D ) CCK8 assay to detect the effect of M2 macrophages on the viability of NCI-H209 cells. ( E ) Flow cytometry to analyze the cell cycle distribution of NCI-H209 cells treated with M2 macrophages. ( F ) Flow cytometry to analyze the cell apoptosis of NCI-H209 cells treated with M2 macrophages. ( G ) Transwell assays to examine the effect of M2 macrophages on the migration of NCI-H209 cells. Scale bar = 100 μm. ( H ) ELISA assay to detect the effect of M2 macrophages on TGF-β expression. ** p < 0.01, *** p < 0.001, ns” stands for “no significant difference

Article Snippet: Subsequently, the cells were incubated with THP-1 cell-specific culture medium (CM-0233, Procell, Wuhan, China) containing IL-4 (20 ng/mL) (HY-P700130AF, MCE, Shanghai, China) and IL-13 (5 ng/mL) (HY-P70568, MCE, Shanghai, China) for 48 h to polarize them into M2 macrophages.

Techniques: CCK-8 Assay, Flow Cytometry, Migration, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Killing efficiency of different physicochemical treatments on recombinant L. lactis strains displaying different binders of proinflammatory cytokines on their surface. The killing efficiency was determined by culturing and counting colonies on agar plates. Complete killing of bacteria i.e., 0 CFU/mL (no bacterial colonies on agar plates) was considered as 100% efficiency. pSD-ZIL6, L. lactis displaying anti-IL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; pNZ8148, L. lactis harboring empty plasmid. The results presented are means ± standard deviation (SD) of three technical replicates from a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Recombinant, Bacteria, Plasmid Preparation, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Cytokine binding ability of recombinant L. lactis bacteria displaying different binders of proinflammatory cytokines on their surface upon exposure to bacteria-killing treatments. ELISA-determined concentrations of recombinant IL6 (A) , TNF (B) , IL17 (C) , and IL8 (D) that remained in the solution following incubation with the corresponding strain of bacteria before and after treatment with heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. The results are presented as means ± SD of three technical replicates of a representative experiment. ns, p = 0.09; *, p ≤ 0.05; **, p = 0.002; ***, p < 0.001 (unpaired two-tailed t -test).

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Incubation, Sonication, Irradiation, Control, Plasmid Preparation, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Determination of binding affinity of ZIL6-displaying L. lactis for human IL6. A constant number of 8 × 10 6 CFU bacteria was incubated with increasing concentrations of human IL6 for 2 h. MFI values were measured by flow cytometry and binding curves were fitted to the data using the Hill equation in the GraphPad Prism v.10.3.1. The dissociation constants (Kd) values were calculated from the binding curves for non-treated, live ZIL6-displaying L. lactis bacteria and bacteria treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. Data are means ± SD of three technical replicates from a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Bacteria, Incubation, Flow Cytometry, Sonication, Irradiation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Quantification of the maximum binding capacity of ZIL6-displaying L. lactis . Increasing concentrations of recombinant human IL6 (0.45, 4.5, 45 and 450 ng) were incubated with a constant number of bacteria (4.5 × 10 7 CFU equivalent to 0.1 mg dry cell weight) in 450 μl. Residual IL6 in the supernatants was quantified by ELISA for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. The percentage of bound IL6 was calculated from the difference measured in the presence of ZIL6-displaying L. lactis and control bacteria (carrying empty plasmid pNZ8148). The data are means ± SD of two biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test).

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Recombinant, Incubation, Bacteria, Enzyme-linked Immunosorbent Assay, Sonication, Irradiation, Control, Plasmid Preparation, Concentration Assay, Comparison

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: (A) Inhibition of STAT3 signaling in HEKblue-IL6R cells by non-viable ZIL6-displaying L. lactis bacteria in comparison to live non-treated strain. ZIL6-displaying L. lactis cells (1 × 10 8 CFU/mL or 1 × 10 9 CFU/mL) were preincubated with IL6 (1 ng/mL) for 2 h. After removal of bacterial cells, the cell-free supernatant containing remainder of IL6 was added to HEKblue-IL6R cells to induce the reporter system. The percentage of STAT3 inhibition was calculated relative to the STAT3 signaling induced by IL6 in the absence of bacteria. The inhibition of STAT3 signaling was determined for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. An anti-IL6 monoclonal antibody (Ab) was used as a positive control. The data are means ± SD of four biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test). (B) The effect of lactic acid production by L. lactis on viability of HEKblue-IL6R cells. HEKblue-IL6R cells (100 000 cells/well) were incubated with ZIL6-displaying L. lactis (2 × 10 7 bacteria/well) for 6, 12, and 24 h, and the viability of HEKblue-IL6R cells was determined with trypan blue. The data are means ± SD of three technical replicates of a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Inhibition, Bacteria, Comparison, Sonication, Irradiation, Control, Plasmid Preparation, Positive Control, Concentration Assay, Incubation

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: IL12B is upregulated in CCR2-deficient hearts and activates autophagy through PI3K/Akt/mTOR pathway inhibition. (A) Western blot results of IL12A and IL12B protein in heart from WT and CCR2 -/- mouse (n = 6). (B) ELISA quantification of IL12A and IL12B levels in heart lysates from WT and CCR2 -/- mouse (n = 6). (C) Western blot results of IL12B protein in BMDMs differentiated from WT and CCR2 -/- mouse (n = 3 independent experiments). (D) Immunofluorescence co-staining of IL12B (green) and the macrophage marker CD68 (red) in heart sections from WT and CCR2 -/- mouse. Scale bar: 5 μm. (E) Western blot results of autophagy markers (LC3B-II and P62) in H9C2 cells treated with recombinant IL12B protein (n = 3 independent experiments). (F) Quantitative PCR analysis of autophagy-related genes Atg12, Atg5, Atg7, Atg3, and Gabarapl1 in H9C2 cells following vehicle or IL12B treatment (2 ng/mL) (n = 3 independent experiments). (G) Western blot results of autophagy-related signaling pathway in H9C2 treated with vehicle or IL12B (2 ng/mL) (n = 3 independent experiments). (H) Western blot results of pPI3K, PI3K and autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in H9C2 treated with vehicle, IL12B (2 ng/mL) alone, or in combination with the PI3K activator 740 Y-P (3 μM) (n = 3 independent experiments).

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Marker, Recombinant, Real-time Polymerase Chain Reaction

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: IL12B promotes cardiomyocyte autophagy in vitro . (A) Cell viability assay of H9C2 treated with DOX (1 μM) in the presence or absence of recombinant IL12B proteins (2 ng/mL) (n = 3 independent experiments). (B) Western blot analysis of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in the same groups (n = 3 independent experiments). (C) Immunofluorescence staining and quantitative analysis of LC3B puncta in the same groups (n = 3 independent experiments). Scale bar: 20 μm. (D) Tandem fluorescence microscopy of H9C2 cells expressing the mRFP-GFP-LC3 reporter. Yellow puncta represent autophagosomes, and red puncta represent autolysosomes. Scale bar: 10 μm (n = 3 independent experiments). (E) Transmission electron microscopy of H9C2. Arrows indicate autolysosomes. Scale bar: 2 μm, 0.5 μm (n = 3 independent experiments).

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: In Vitro, Viability Assay, Recombinant, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy, Expressing, Transmission Assay, Electron Microscopy

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: Recombinant IL12B administration protects against DIC through autophagy activation in vivo . (A) Experimental design for in vivo IL12B administration. (B) Representative M-mode echocardiographic tracings of mouse from the vehicle, DOX alone, and DOX + IL12B groups. Scale bar: 0.1 s, 2 mm. (C, D) Quantitative analysis of EF and FS in the indicated groups (n = 6). (E–G) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (H) Western blot analysis of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from the same groups (n = 6). (I) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (J) Immunofluorescence staining and quantitative analysis of LC3B puncta in cardiomyocytes from the same groups. Scale bar: 50 μm (n = 6). (K) Transmission electron microscopy analysis of autolysosome density from the same groups. Arrows indicate autolysosomes. Scale bars: 1 μm and 0.5 μm (n = 6).

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: Recombinant, Activation Assay, In Vivo, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: IL12B neutralization abolishes CCR2 deficiency-mediated cardioprotection in vivo . (A) Experimental design of IL12B neutralization in CCR2 -/- mouse. (B) Representative M-mode echocardiographic tracings of CCR2 -/- mouse treated with vehicle, DOX alone, or DOX + anti-IL12B Neu. Scale bar: 0.1 s, 2 mm. (C, D) Quantitative results of EF and FS in the indicated groups (n = 6). (E–G) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (H) Western blot results of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from CCR2 -/- mouse treated with vehicle, DOX alone, or DOX + anti-IL12B Neu (n = 6). (I) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (J) Immunofluorescence staining and quantitative analysis of LC3B puncta in cardiomyocytes from the same groups. Scale bar: 50 μm (n = 6). (K) Transmission electron microscopy analysis of autolysosome density in cardiomyocytes from the same groups. Arrows indicate autolysosomes. Scale bars: 1 μm and 0.5 μm (n = 6).

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: Neutralization, In Vivo, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: Pharmacological CCR2 inhibition protects against DIC without compromising the antitumor efficacy of doxorubicin in 4T1 breast cancer model. (A) Cell viability assay of 4T1 breast cancer cells treated with DOX or in combination with CCR2 antagonist4 (n = 3 independent experiments). (B, C) Tumor growth curves and representative tumor images at the study endpoint in a syngeneic 4T1 breast cancer model. Scale bar: 1 cm (n = 6). (D) Representative M-mode echocardiographic tracings of tumor bearing mouse treated with vehicle, DOX alone, CCR2 antagonist4 alone or DOX + CCR2 antagonist4. Scale bar: 0.1 s, 2 mm. (E, F) Quantitative results of EF and FS in the same groups (n = 6). (G–I) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (J) Western blot results of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from the same groups (n = 6). (K) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (L) Working model. DOX administration triggers cardiomyocyte dysfunction, ultimately leading to heart failure. In contrast, loss of function of CCR2 initiates a protective cascade. Specifically, CCR2 deficiency increases the secretion of IL12B. This cytokine directly targets cardiomyocytes, suppressing PI3K/Akt/mTOR signaling to trigger protective autophagy, which clears damaged organelles and toxic proteins, thus sustaining cellular homeostasis and cardiac function.

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: Inhibition, Viability Assay, Western Blot, Real-time Polymerase Chain Reaction