Journal: Nature Communications

Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma

doi: 10.1038/s41467-025-65056-9

Figure Lengend Snippet: a Schematic illustration of the induction of M2 TAMs, the 1-6/TAM spheroids formulation, the transwell co-incubation, and their respective treatments. b Western blot analysis of GPX4 and FSP1 expression in M2 TAMs after different treatments. The experiment was repeated three times independently with similar results. Uncropped blots in Source Data. c Quantification of grayscale intensity of GPX4 and FSP1 in ( b ) ( n = 3 independent experiments). Protein expression levels in PBS group were normalized to 1. d Live/dead staining of M2 TAMs after different treatments (Scale bar = 200 μm). Viable cells were stained with calcein-AM (green), and dead cells were stained with PI (red). The experiment was repeated three times independently with similar results. e Quantification of the percentage of viable and dead M2 TAMs in ( d ) ( n = 3 independent experiments). f Flow cytometric analysis of Annexin V-FITC/propidium iodide (PI)-stained M2 TAMs (gated on F4/80 + CD206 + macrophages) after different treatments. g Quantification of the percentage of apoptotic cells in ( f ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. h The cell proliferation of M2 TAMs after different treatments measured by CCK8 ( n = 3 independent experiments). The concentrations of SF and vF contained in nanoparticles were both 3 μM. i Fluorescence images of C11 BODIPY 581/591 -stained M2 TAMs after different treatments (Scale bar = 100 μm). The experiment was repeated three times independently with similar results. j Fluorescence images of live/dead stained 1-6/TAM spheroids after different treatments (Scale bar = 500 μm). Viable cells were stained with calcein-AM (green), and dead cells were stained with PI (red). Z-stack scanning of the spheroids was performed with slices distanced by 14.36 μm. The experiment was repeated three times independently with similar results. k Flow cytometric analysis of anti-CRT-stained Hepa1-6 cells after different treatments. l Quantification of the percentage of CRT positive cells in ( k ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. m The ATP assay of Hepa1-6 cells after different treatments ( n = 3 independent experiments). n The HMGB1 assay of Hepa1-6 cells after different treatments ( n = 3 independent experiments). o Flow cytometric analysis of anti-CD80/CD86-stained BMDCs (gated on CD11c + DCs) after different treatments. p Quantification of the percentage of mature DCs in ( o ) ( n = 3 independent replicates). The experiment was repeated twice independently with similar results. q The assay of TNF, IL-6, and IL-12 in BMDCs after different treatments ( n = 3 independent experiments). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( c , g , h , l – n , p , q ) and P -values were indicated. Source data are provided as a Source Data file. The elements in Fig. 5a were created by Adobe Illustrator.

Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).

Techniques: Formulation, Incubation, Western Blot, Expressing, Staining, Fluorescence, ATP Assay

Journal: Nature Communications

Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma

doi: 10.1038/s41467-025-65056-9

Figure Lengend Snippet: a Body weight of mice with intravenous treatments during a 28-day period ( n = 5 independent mice). b The blood analysis for the liver/kidney functions including ALT, AST, BUN, and CRE was determined on day 28 following the treatments ( n = 5 independent mice). c The curve of injected drug concentration (ID %) versus time point was plotted in subcutaneous HCC mice ( n = 6 independent mice). d Fluorescence imaging of the biodistribution in orthotopic HCC mice at 12 h after different treatments (tumors marked with yellow circles; n = 6 independent mice). e Tumor-to-background ratio for different treatments in ( d ) ( n = 6 independent mice). f Quantitative biodistribution analysis of DiR-labeled nanoparticles in major organs and liver tumors in ( d ) ( n = 6 independent mice). g Fluorescence images of Sv@PM-M2p inside HCC cells, M2 TAMs, M1 TAMs, DCs, and T cells in tumors ( n = 6 independent mice; Scale bar = 100 μm). Sv@PM-M2p was labeled with Rhodamine (red), the nuclei and cell markers (HCC, CK8; M2, CD206; M1, CD86; DC, CD11c; T, CD3) were stained with DAPI (blue) and antibody (green), respectively. h Quantification of the percentage of colocated cells in ( g ) ( n = 6 independent mice). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( c , e , f , h ) and P -values were indicated. Source data are provided as a Source Data file.

Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).

Techniques: Injection, Concentration Assay, Fluorescence, Imaging, Labeling, Staining

Journal: Nature Communications

Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma

doi: 10.1038/s41467-025-65056-9

Figure Lengend Snippet: a Schematic illustration of the research procedure in subcutaneous HCC mouse model. b Relative tumor volume curves of different treatment groups ( n = 5 independent mice). c Animal survival after different treatments ( n = 5 independent mice). d Quantification of fluorescence intensity in Supplementary Fig. f ( n = 5 independent mice). e Immunofluorescence images of tumor tissue slices collected from different groups stained with anti-F4/80/CD206, anti-F4/80/CD86, and anti-CD11c/MHC II after different treatments ( n = 5 independent mice; Scale bar = 100 μm). f Quantification of the percentage of F4/80 + CD206 + M2 TAMs, F4/80 + CD86 + M1 TAMs, and CD11c + MHC II + actDCs in ( e ) ( n = 5 independent mice). g Schematic illustration of the research procedure in orthotopic HCC mouse model. h Relative bioluminescence intensity curves for the various treatment groups ( n = 5 independent mice). i Animal survival after different treatments ( n = 5 independent mice). j Flow cytometric analysis of anti-F4/80/CD206-stained macrophages, anti-F4/80/CD86-stained macrophages, anti-CD11c/MHC II-stained DCs, anti-CD3/CD4-stained T cells, anti-CD3/CD8-stained T cells, and anti-CD4/Foxp3-stained T cells in tumor tissues after different treatments ( n = 5 independent mice). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( b , d , f , h ) or log-rank test ( c , i ) and P -values were indicated. Source data are provided as a Source Data file. The elements in Fig. 7a, g were created by Adobe Illustrator.

Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).

Techniques: Fluorescence, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Co-delivery of sorafenib and an FSP1 inhibitor triggers dual ferroptosis in tumor cells and immunosuppressive macrophages for enhanced immunotherapy in mouse models of hepatocellular carcinoma

doi: 10.1038/s41467-025-65056-9

Figure Lengend Snippet: a Schematic illustration of the research procedure in PDX subcutaneous mouse model. b Relative tumor volume curves of different treatment groups ( n = 5 independent mice). c Collected tumor tissues of mice at the end of treatment in different groups. d Tumor weight at the end of treatment in different groups ( n = 5 independent mice). e Immunofluorescence images of tumor tissue slices collected from different groups stained with Liperfluo after different treatments ( n = 5 independent mice; Scale bar = 100 μm). f Quantification of fluorescence intensity in ( e ) ( n = 5 independent mice). g Flow cytometric analysis of anti-CD68/CD206-stained macrophages (gated on CD45 + cells), anti-CD68/CD80-stained macrophages (gated on CD45 + cells), anti-HLA-DR/CD11c-stained DCs (gated on CD45 + Lin-1 – T cells), and anti-CD3/CD8-stained T cells (gated on CD45 + cells) in tumor tissues after different treatments ( n = 5 independent mice). h Quantification of the percentage of CD68 + CD206 + M2 TAMs, CD68 + CD80 + M1 TAMs, HLA-DR + CD11c + actDCs, and CD3 + CD8 + Tc cells in ( g ) ( n = 5 independent mice). Unless specified otherwise, error bars represent the mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s test ( b , d , f , h ) and P -values were indicated. Source data are provided as a Source Data file. The elements in Fig. 9a were created by Adobe Illustrator.

Article Snippet: PerCP/Cyanine5.5 anti-mouse F4/80 (1:200, #E-AB-F0995J), APC anti-mouse CD206 (1:200, #E-AB-F1135E), PE/Cyanine7 anti-mouse CD86 (1:200, #E-AB-F0994H), Fluor Red 780 anti-mouse CD80 (1:200, #E-AB-F0992S), APC anti-mouse CD11c (1:200, #E-AB-F0991E), Fluor Violet 450 anti-mouse CD3 (1:200, #E-AB-F1013Q), Fluor Red 780 anti-mouse CD4 (1:200, #E-AB-F1097S), PerCP/Cyanine5.5 anti-mouse CD8 (1:200, #E-AB-F1104J), FITC anti-human/mouse CD44 (1:200, #E-AB-F1100C), PE anti-mouse Foxp3 (1:200, #E-AB-F1238D), FITC anti-mouse MHC II (1:200, #E-AB-F0990C), APC anti-mouse CD62L (1:200, #E-AB-F1011E), APC anti-mouse PD-L1 (1:200, #E-AB-F1132E), PerCP anti-human CD45 (1:200, #E-AB-F1137F), Fluor647 anti-human CD68 (1:200, #E-AB-F1299M), FITC anti-human CD206 (1:200, #E-AB-F1161C), PE anti-human CD80 (1:200, #E-AB-F1232D), APC anti-human HLA-DR (1:200, #E-AB-F1111E), PE anti-human CD11c (1:200, #E-AB-F1118D), APC anti-human CD3 (1:200, #E-AB-F1001E), and FITC anti-human CD8 (1:200, #E-AB-F1110C) were purchased from Elabscience (Wuhan, China).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Journal for immunotherapy of cancer

Article Title: Carbohydrate ligand engagement with CD11b enhances differentiation of tumor-associated myeloid cells for immunotherapy of solid cancers.

doi: 10.1136/jitc-2022-006205

Figure Lengend Snippet: Figure 4 BG34-200-AF647 intravenous administration shows a direct impact on the tumor-associated inflammatory monocytes (TAIMs) in the peripheral blood. (A) Kinetic distribution of BG34-200-AF647 in the peripheral blood of KPC tumor- bearing mice (I and II) and tumor-free mice (III and IV). (I) and (III): MFI of BG34-200-AF647 in plasma samples of mice, as determined by colorimetric analysis. Tumor-free plasma sample spiked with BG34-200-AF647 (0.1 mg/mL) served as positive control. MFI of plasma sample from individual mouse was graphed as mean±SD. **, p < 0.01. (II) and (IV): Frequency of BG34-200-AF647+ cells in the WBCs, as determined by FACS analysis. (B) Surface expression of Ly6C and Ly6G by the CD11b+BG34-200-AF647+ cells in the peripheral blood in the KPC tumor-bearing mice at 10 hours after BG34-200-AF647 intravenous administration, as determined by FACS. (C) Frequency of TAIMs and the BG34-200-AF647+ TAIMs in the peripheral blood in the KPC, B16F10, K7M2 tumor-bearing and tumor-free mice. (D) Plasma CCL2 concentrations in mice bearing different solid tumors with PBS or BG34-200-AF647 treatment, as determined by ELISA assays. Tumor-free mouse plasma served as controls. (E) MFI of BG34-200-AF647 in tumors, tumor draining lymph nodes (TdLNs), and different organs, as determined by colorimetric analysis. MFI of samples from mice were graphed as mean±SD. Two of the three data points represent pooled samples from two mice and one data point represents sample from one mouse. (F and G) Surface expression of CD11c, MHC II, and CCR2 by the KPC tumor-infiltrating CD11b+BG34-200-AF647+ cells (F) and circulating CD11b+BG34-200-AF647+ cells (G), as determined by FACS. For A, at 2, 10, 24 and 72 hours after BG34-200-AF647 intravenous injection, blood samples were collected and processed to collect white blood cells. The cells were then analyzed by FACS to determine the frequency of the BG34-200-AF647+ cells. Frequency of cells from individual mouse were graphed as mean±SD. For B, C, D, and G, the plasma and WBCs were collected at 10 hours after BG34-200-AF647 intravenous administration. For F, the tumor-infiltrating cells were collected at the 24 hours after BG34-200-AF647 intravenous administration. Mice bearing day-5 KPC tumors were intravenously administered with PBS or AF647-tagged BG34-200 (BG34-200-AF647) at 50 mg/kg. At 20 min, 2, 4, 10, 24 and 72 hours after BG34-200-AF647 injection, mice were sacrificed to harvest blood, tissue, organs, and tumor samples. n=5. BM, bone marrow; CTL, control; FACS, fluorescence-activated cell analysis; IV, intravenous; MFI, mean fluorescence intensity; PBS, Phosphate- Buffered Saline; WBCs, white blood cells.

Article Snippet: Following the culture period, we surface stained the cells with anti- CD11c- PE (R&D Systems), fixed, permeabilized, and intracellular stained them using anti- CD11b- AF488 antibodies (BG Biosciences), followed by FACS analysis.

Techniques: Clinical Proteomics, Positive Control, Expressing, Enzyme-linked Immunosorbent Assay, Injection, Control, Fluorescence, Cell Analysis, Saline

Journal: Journal for immunotherapy of cancer

Article Title: Carbohydrate ligand engagement with CD11b enhances differentiation of tumor-associated myeloid cells for immunotherapy of solid cancers.

doi: 10.1136/jitc-2022-006205

Figure Lengend Snippet: Figure 6 The intravenous administration of BG34-200 can promote the differentiation of TAIMs into mo-DCs, resulting in an increased capacity for antigen presentation. In panel (A and B): FACS sorting of CD11b+CCR2+ cells from the peripheral blood of mice with different solid tumors (KPC, B16F10, and K7M2) treated with PBS (A) or BG34-200 (B) at 10 hours following the intravenous administration, and RT-PCR analysis of the relative mRNA expression of PU.1 and MafB in the sorted cells. Cells were sorted from blood of mice bearing three different solid tumors (KPC, B16F10, and K7M2). Mouse BM derived inflammatory monocytes (PMA treatment at 10 ng/mL) served as control (CTL). The mRNA levels were expressed relative to the normalized CTL. In B, the mean value of PU.1 expression in the sorted cells from KPC, B16F10 and K7M2 mice treated with PBS is represented by the blue, green, and red dashed lines, respectively. In A and B, n=5; 88, p<0.01; ***, p<0.001. (C) FACS analysis of the frequency of CD11c+ cells in mature mo-DCs derived from the blood of KPC mice treated with PBS or BG34-200. (D) The percentage of proliferating OT I CD8+ T cells in response to DCs pulsed by irradiated B16F10-OVA cells, as determined by FACS. In C and D, the cells sorted from PBS or BG34-200 groups were cultured in DC medium supplemented with GM-CSF/ IL-4 and stimulated by TNF-α to generate mature mo-DCs. The mo-DCs were then pulsed by B16F10-OVA cells at 1:4 ratio and cultured with OT I CD8+ T cells at CD11c+ cells:T cells ratio of 5:1. At the end of culture, cells were stained by anti-CD8- FITC and ki67, followed by FACS analysis to assess T-cell proliferation. BM, bone marrow; FACS, fluorescence-activated cell analysis; FSC, Forward Scatter; GM-CSF, Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF); IL, interleukin; mo- DCs, monocyte-derived dendritic cells; mRNA, messenger RNA; PBS, Phosphate-Buffered Saline; PMA, phorbol 12-myristate- 13-acetate; RT-PCR, real-time PCR; TAIMs, tumor-associated inflammatory monocytes; TNF, tumor necrosis factor.

Article Snippet: Following the culture period, we surface stained the cells with anti- CD11c- PE (R&D Systems), fixed, permeabilized, and intracellular stained them using anti- CD11b- AF488 antibodies (BG Biosciences), followed by FACS analysis.

Techniques: Immunopeptidomics, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Control, Irradiation, Cell Culture, Staining, Fluorescence, Cell Analysis, Saline, Real-time Polymerase Chain Reaction

Journal: Journal for immunotherapy of cancer

Article Title: Carbohydrate ligand engagement with CD11b enhances differentiation of tumor-associated myeloid cells for immunotherapy of solid cancers.

doi: 10.1136/jitc-2022-006205

Figure Lengend Snippet: Figure 7 BG34-200 treatment modulates function of the TAIMs-derived DCs through triggering internalization of binding complexes, phagocytosis, F-actin cytoskeletal rearrangement, and intrinsic ICAM-1 surface aggregation. (A) The per cent expression of DC-related biomarkers by matured CD11c+ cells derived from KPC tumor-bearing mice treated with PBS or BG34-200 treatment, as determined by FACS. Within the CD11b+ cells, a fraction of cells of the BG34-200-treated group exhibited lower fluorescence intensity than the cells of the PBS-treated group (red rectangles). Within CD54+ cells, a fraction of cells of the BG34-200-treated mice showed higher fluorescence intensity than the cells of the PBS-treated group (green rectangle frame). (B) Schematics of the ex vivo experimental setting to study the internalization rates of binding complexes and the phagocytosis rates. KPC-derived cell line TB32048 was directly injected into pancreas to create the KPC-tumor-bearing mice. For both assays, the TAIM-derived immature DCs were co-cultured with BG34 samples for 30, 60, 90, 120, 150 and 180 min. (C) Averaged MFI of the single CD11c+CD11b-AF488+BG34-AF647+ cells at different co-culture time, as determined by FACS. (D) Average MFI of the single CD11c-PE+E coli –AF488+ at different co-culture time, as determined by FACS. For C and D, the averaged MFI of the single cells = (Total MFI of the positive cells) / (total number of the positive cells); cells of PBS, BG34-50, BG34-200, and BG34-500 groups were examined in triplicates; Data expressed as mean±SD. (E) Stimulated emission depletion (STED) microscopic imaging (100×) of mature DCs from PBS and BG34 groups. The DCs were stained with ICAM-1- AF488 and F-actin-CF568 and fixed on non-functional glass surface. For statistical analysis, the STED imaging was performed on 50 randomly picked cells of each group. Image analysis of the four groups of 50 cells resulted in the identification of four different status (I, II, III, and IV) (left). The numbers of cells adopting each status were recorded for PBS, BG34-50, BG34-200, and BG34-500 groups to assess the effect of different treatments on ICAM-1 status and F-actin cytoskeletal structure (right). Abs, antibodies; DCs, dendritic cells; E. coli; Escherichia coli; FACS, fluorescence-activated cell analysis; GM-CSF, Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF); ICAM-I, intercellular adhesion molecule I; IV, intravenous; MFI, mean fluorescence intensity; PBS, Phosphate-Buffered Saline; ROI, region of interest; TAIMs, tumor-associated inflammatory monocytes.

Article Snippet: Following the culture period, we surface stained the cells with anti- CD11c- PE (R&D Systems), fixed, permeabilized, and intracellular stained them using anti- CD11b- AF488 antibodies (BG Biosciences), followed by FACS analysis.

Techniques: Derivative Assay, Binding Assay, Expressing, Fluorescence, Ex Vivo, Injection, Cell Culture, Co-Culture Assay, Imaging, Staining, Functional Assay, Cell Analysis, Saline

Journal: Journal of Neuroinflammation

Article Title: The effect of dipeptidyl peptidase IV on disease-associated microglia phenotypic transformation in epilepsy

doi: 10.1186/s12974-021-02133-y

Figure Lengend Snippet: DPP4 participates in DAM phenotypic transformation in epileptic mice. a – c Gene expression in hippocampus from mice treated with kainic acid or kainic acid + sitagliptin ( n = 6–7 per group). Selected markers: DAM: Itgax and Axl ; homeostatic: Cx3cr1 . d – f Representative western blotting images and statistical analysis of DPP4 and ITGAX in the hippocampus ( n = 4). β-Actin was used as a loading control. g Representative overlay images of IBA1/ITGAX/DAPI staining in the hippocampus (IBA1, red; ITGAX, green; DAPI, blue). Scale bar = 25 μm. h The statistical analysis of ITGAX in IBA1 + microglia is shown on the left side. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Primary antibodies against the following proteins were used in the experiments: DPP4 (1:1000, Abcam), CD68 (1:1000, Abcam), ITGAX (1:1000, Proteintech), NF-κB p65 (1:1000, Cell Signaling Technology, USA), MyD88 (1:1000, Proteintech), TRAF6 (1:3000, Proteintech) and NF-κB phosphorylated p65 (p-p65; 1:1000, Cell Signaling Technology, USA).

Techniques: Transformation Assay, Expressing, Western Blot, Staining

Journal: Journal of Neuroinflammation

Article Title: The effect of dipeptidyl peptidase IV on disease-associated microglia phenotypic transformation in epilepsy

doi: 10.1186/s12974-021-02133-y

Figure Lengend Snippet: Inhibition of DPP4 alters microglial morphology and function in vitro. a Flowchart of the experiment in vitro. b – d Whole-cell lysates were subjected to western blotting to examine the protein levels of ITGAX and DPP4 in BV2 microglial cells after treatment with LPS or LPS + sitagliptin. Data were normalized to β-actin ( n = 3–4). e Representative morphology of BV2 microglia in each group under a phase-contrast microscope. Scale bar = 50 μm. f Statistics of processes number, g soma area and h maximum length in BV2 microglia ( n = 20–24 cells per group). i , j Microglial migration was measured using wound healing assays. Scale bar = 200 μm. Data are presented as the mean ± SEM. * P < 0.05, *** P < 0.001

Article Snippet: Primary antibodies against the following proteins were used in the experiments: DPP4 (1:1000, Abcam), CD68 (1:1000, Abcam), ITGAX (1:1000, Proteintech), NF-κB p65 (1:1000, Cell Signaling Technology, USA), MyD88 (1:1000, Proteintech), TRAF6 (1:3000, Proteintech) and NF-κB phosphorylated p65 (p-p65; 1:1000, Cell Signaling Technology, USA).

Techniques: Inhibition, In Vitro, Western Blot, Microscopy, Migration