Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: Effect of FGF2 deficiency on BMDM apoptosis and polarization. a – c FGF2 deletion increased BMDM apoptosis. a Apoptosis in BMDM deprived of FBS for 24 h was assessed by flow cytometry ( n = 4). b - c Percentage of PI + Annexin V + and PI- Annexin V + BMDM after starvation. d - k FGF2 deletion in BMDM promoted M1 polarization. d - g Flow cytometric analysis of macrophage markers in BMDM treated with LPS or IL4, including CD86, iNOS, CD206, and Arg1 ( n = 3). h - k The levels of CD86, iNOS, CD206 and Arg1 in BMDM after treatment with LPS or IL4. N represents no treatment; * p < 0.05, vs. WT; Ψ p < 0.05, vs. N + WT; Ω p < 0.05, vs. N + FGF2 KO

Article Snippet: The tissue sections were incubated overnight at 4°C with primary antibodies, including the rabbit polyclonal antibody CD206 (1:200, Cat No. A02285-2, Boster, Wuhan, China), rabbit monoclonal antibody CD86 (1:100, Cat No. BM4121, Boster, Wuhan, China), rabbit polyclonal antibody F4/80 (1:100, Cat No. 29414-1-AP, Proteintech, Wuhan, China), anti-mouse NLRP3 (1:200, Cat No.68102-1-Ig, Proteintech, Wuhan, China), Caspase-1 p20 Rabbit pAb (1:400, Cat No.bs-10743R, Bioss, Beijing, China) and ASC/TMS1 Rabbit PolyAb (1:200, Cat No.10500-1-AP, Proteintech, Wuhan, China).

Techniques: Flow Cytometry

Journal: Molecular Biomedicine

Article Title: Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion

doi: 10.1186/s43556-024-00203-0

Figure Lengend Snippet: Mice reconstituted with FGF2 KO macrophages and subjected to CLP demonstrate increased M1 polarization in lung tissue. a - f The presence and levels of CD206, CD86, and F4/80 markers on macrophages within lung tissue were identified and quantitatively assessed using immunofluorescence staining. Bar is 20 μm. * p < 0.05, vs. WT; Δ p < 0.05 vs. WT + LPS; # p < 0.05 vs. FGF2 KO

Article Snippet: The tissue sections were incubated overnight at 4°C with primary antibodies, including the rabbit polyclonal antibody CD206 (1:200, Cat No. A02285-2, Boster, Wuhan, China), rabbit monoclonal antibody CD86 (1:100, Cat No. BM4121, Boster, Wuhan, China), rabbit polyclonal antibody F4/80 (1:100, Cat No. 29414-1-AP, Proteintech, Wuhan, China), anti-mouse NLRP3 (1:200, Cat No.68102-1-Ig, Proteintech, Wuhan, China), Caspase-1 p20 Rabbit pAb (1:400, Cat No.bs-10743R, Bioss, Beijing, China) and ASC/TMS1 Rabbit PolyAb (1:200, Cat No.10500-1-AP, Proteintech, Wuhan, China).

Techniques: Immunofluorescence, Staining

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Sanguinarine, identified as a natural alkaloid LSD1 inhibitor, suppresses lung cancer cell growth and migration

doi: 10.22038/IJBMS.2022.62541.13851

Figure Lengend Snippet: Sanguinarine increased cellular levels of H3K4me2 and H3K9me2 and up-regulated the expression of CD86 in NSCLC cell lines. (A) and (B) Expression of H3K4me2 and H3K9me2 in H1975 and H1299 cells treated with sanguinarine for 48 hr, respectively, with H3 as loading control; (C) and (D) The expression of CD86 was detected, GADPH was used as a loading control. Data are the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01, *** P <0.005

Article Snippet: Antibodies used were against GAPDH (GOOD HERE, AB-P-R 001), H3 (HUABIO, EM30605), H3K4me2 (HUABIO, R1110-3), H3K9me2 (HUABIO, ET1611-51), CD86 (BOSTER, BM4121), E-Cadherin (HUABIO, ET1607-75), N-Cadherin (HUABIO, ET1607-37), Bax (HUABIO, ER0907), Bcl-2 (HUABIO, ER1082-97).

Techniques: Expressing, Control

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: ILT4 in EGFR-activated tumor cells promoted TAM recruitment and M2-like polarization. (A-B) Patients with high ILT4 expression in tumor cells of NSCLC tissues showed markedly elevated infiltration of CD68 + TAMs by IHC analysis. (A) Representative images of ILT4 expression and TAM infiltration, brown granules represent positive staining. (B) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 (median) and ≥ 6. Scale bar: 20 µm. (C-D) TAMs induced by PC9 and H1975 cells displayed increased migration ability compared with parental macrophages, whereas ILT4 knockdown in PC9 and H1975 cells restricted tumor-induced TAM migration. Tumor cells were first transfected with lentivirus carrying ILT4 shRNA for 48 h, and CM was collected for macrophage culture and TAM induction. The migration ability of TAMs was evaluated by the Transwell migration assay. (C) Images of migrated cells and (D) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. ### , p < 0.001 compared with the LV-shNC group. (E-F) ILT4 knockdown in PC9 and H1975 cells decreased the secretion of CCL2 and CCL5. (E) showed ELISA results of CCL2, (F) showed CCL5. (G-H) Recombinant human CCL2 or CCL5 reversed ILT4 knockdown and decreased macrophage migration. 100 ng/mL recombinant human CCL2 or 200 ng/mL CCL5 were added to the CM of ILT4 knockdown PC9 and H1975 cells to induce TAM migration. The migration ability of TAMs was evaluated by the Transwell migration assay. (G) Images of migrated cells. (H) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. compared with LV-shNC group. (I-J) Patients with high ILT4 expression in tumor cells displayed more frequent CD206 + but less CD86 + TAM accumulation in tumor tissues by IHC analysis. (I) Representative images of CD68 + TAMs, CD206 + M2-like TAMs and CD86+ M1-like TAMs frequencies; brown granules represent positive staining. (J) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 and ≥ 6 respectively. Scale bar: 20 µm. (K-L) ILT4 knockdown in PC9 and H1975 cells decreased M2-like markers (CD163, CD206, IL-10 and Arg1) and increased M1-like markers (CD80, CD86, IL-12 and TNFα) in TAMs by real-time PCR (K) and flow cytometry (L). TAMs were induced by CM as in (D) for 24 h. (M-N) TAMs induced by ILT4-downregulated PC9 and H1975 cells promoted the proliferation (M) and IFN-γ expression (N) of T cells. For T cell proliferation assay, CD3 + T cells separated from fresh PBMCs were first pre-activated by anti-CD3 for 24 h and stained with CFSE (1:1000), and then co-cultured for 4 days with TAMs induced by CM of ILT4-downregulated PC9 and H1975 cells. Flow cytometry was performed to determine CFSE strength. For IFN-γ expression, pre-activated CD3 + T cells were co-cultured with TAMs as described above for 48 h and IFN-γ levels were analyzed by flow cytometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Arg1: arginase 1; CM, conditioned medium; LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA.

Article Snippet: Following antigen retrieval, the sections were incubated with 3% hydrogen peroxide for 20 min. Tissue slides were then incubated overnight at 4 °C with following primary antibodies: anti-ILT4 antibody (1:50; Affinity; Cat No. DF9604), anti-pEGFR antibody (1:200; Abcam; Cat No. ab40815), anti-PD-L1 antibody (1:100; CST; Cat No.13684), anti-CD68 antibody (1:100; Abcam; Cat No. ab125212), anti-CD206 antibody (1:100; Abcam; Cat No. ab64693), anti-CD163 antibody (1:100; Abcam; Cat No. ab182422), anti-CD3 antibody (1:200; Abcam; Cat No. ab5690; 1:100; CST; Cat No. 85061), anti-IFN-γ antibody (1:50; Affinity; Cat No. DF6045), and anti-F4/80 antibody (1:200; Abcam; Cat No. ab111101), anti-CD80 antibody (1:500; Boster; Cat No. BM4121), anti-CD86 antibody (1:500; Proteintech; Cat No. 66406-1-Ig), anti-PIRB antibody (1:100; Immunoway; Cat No. YN1914).

Techniques: Expressing, Staining, Migration, Knockdown, Transfection, shRNA, Transwell Migration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Real-time Polymerase Chain Reaction, Flow Cytometry, Proliferation Assay, Cell Culture, Control

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: PIR-B and PD-L1 blockade synergistically prevented tumor growth and immune escape in vivo . (A-B) PIR-B knockdown or PD-L1 neutralization markedly inhibited transplanted tumor growth in C57BL/6 mice, while combined PIR-B and PD-L1 blockade yielded the slowest tumor growth rate. Mouse lung cancer cell line LLC (2×10 5 cells/mouse) infected with both EGFR overexpression and PIR-B knockdown- or control lentivirus was subcutaneously inoculated into 6-8-week female C57BL/6 mice. PD-L1 inhibitory antibody or control IgG (200 µg) were intraperitoneally injected into the tumor-bearing mice every 4 days from day 7 post-tumor inoculation. Tumor sizes were measured every 4 days and presented as mean ± SD (n = 6 mice/group). (A) Tumor growth rate in each group and (B) Tumor images obtained from each mouse. Scale bar: 2 cm. (C) Tumors in PIR-B knockdown or PD-L1 inhibition groups showed significantly smaller tumor weight than the control group, while tumors in combined PIR-B and PD-L1 blockade groups showed the smallest tumor weight. The histogram shows the mean ± SD of tumor weights from each group at the endpoint of the experiments (n = 6 mice/group). (D) F4/80 + macrophages in CD45 + leukocytes were remarkably increased in spleens of tumor-bearing mice compared with normal mice. However, when tumors were treated with PIR-B knockdown lentivirus or/and PD-L1 antibody, F4/80 + macrophages were markedly decreased with the lowest number in the combined PIR-B and PD-L1 blockade group. The proportions of F4/80 + macrophages in CD45 + leukocytes were determined by flow cytometry. (E) Blocking PIR-B or PD-L1 remarkably decreased TAMs in tumor tissues, while combined PIR-B and PD-L1 blockade displayed the lowest number of TAMs. The accumulation of TAMs in mouse tumor tissues was evaluated by IHC analyses. (F) CD206 + macrophages in spleens were remarkably induced by tumor inoculation. However, when PIR-B or PD-L1 were blocked using specific knockdown lentivirus or neutralizing antibody, CD206 + macrophages were significantly reduced. The combined blockade of both molecules yielded the lowest CD206 + macrophage numbers in spleens. CD206 + macrophages in leukocytes were determined by flow cytometry. (G-H) PIR-B or PD-L1 inhibition markedly decreased CD206 + TAMs but increased CD86 + TAMs in transplanted tumor tissues, and combination therapy generated the lowest frequency of CD206 + TAMs but highest frequency of CD86+ TAMs in tumors. The accumulation of CD206 + TAMs (G) and CD86 + TAMs (H) was determined by IHC analysis. (I) The spleens and blood of tumor bearing mice showed much less CD3 + T cell proportion relative to normal mice. However, when PIR-B and/or PD-L1 were inhibited, the decreased proportion of CD3 + T cells in CD45 + lymphocytes was significantly reversed with the highest CD3 + T cell frequency in combined blockade group both in spleens and blood. The frequency of CD3 + T cells in CD45 + lymphocytes was detected using flow cytometry. (J) Tumor tissues from PIR-B-downregulated or anti-PD-L1-treated mice showed increased CD3 + T cell numbers in tumor tissues. Combined inhibition of both molecules generated the highest enrichment of CD3 + T cells as indicated by IHC analysis. (K-L) T cells in either PIR-B or PD-L1 blockade group displayed increased IFN-γ expression, while the combination therapy group showed the highest IFN-γ levels. IFN-γ expression in mouse spleens and blood (K) was determined by flow cytometry and in tumors (L) by IHC. (M-O) Knockdown of PIR-B in LLC inhibited tumor growth, and decreased tumor sizes and weights in immunodeficient NSG mice. Cell preparation, cell injection numbers and procedures were identical as described in (A). Tumor sizes were measured every 4 days (n = 7 mice/group). (M) Tumor growth rate in each group and (N) Tumor images from each mouse. Scale bar: 2 cm. (O) Tumors in PIR-B knockdown group showed smaller tumor weight than the control group. Data in (M) and (O) are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Anti: inhibitory antibody; LV-shPIR-B: lentivirus carrying PIR-B shRNA; LV-shNC: lentivirus carrying control shRNA.

Article Snippet: Following antigen retrieval, the sections were incubated with 3% hydrogen peroxide for 20 min. Tissue slides were then incubated overnight at 4 °C with following primary antibodies: anti-ILT4 antibody (1:50; Affinity; Cat No. DF9604), anti-pEGFR antibody (1:200; Abcam; Cat No. ab40815), anti-PD-L1 antibody (1:100; CST; Cat No.13684), anti-CD68 antibody (1:100; Abcam; Cat No. ab125212), anti-CD206 antibody (1:100; Abcam; Cat No. ab64693), anti-CD163 antibody (1:100; Abcam; Cat No. ab182422), anti-CD3 antibody (1:200; Abcam; Cat No. ab5690; 1:100; CST; Cat No. 85061), anti-IFN-γ antibody (1:50; Affinity; Cat No. DF6045), and anti-F4/80 antibody (1:200; Abcam; Cat No. ab111101), anti-CD80 antibody (1:500; Boster; Cat No. BM4121), anti-CD86 antibody (1:500; Proteintech; Cat No. 66406-1-Ig), anti-PIRB antibody (1:100; Immunoway; Cat No. YN1914).

Techniques: In Vivo, Knockdown, Neutralization, Infection, Over Expression, Control, Injection, Inhibition, Flow Cytometry, Blocking Assay, Generated, Expressing, shRNA

Journal: Scientific Reports

Article Title: Identification of m6A methyltransferase-related WTAP and ZC3H13 predicts immune infiltrates in glioblastoma

doi: 10.1038/s41598-025-88671-4

Figure Lengend Snippet: Relative expression of genes expression in GBM tissues. Expression levels of WTAP and ZC3H13 genes were compared with those of the control group on GBM tissues, 10 GBM and adjacent tissues, respectively ( A ). The expression levels of siRNA WTAP and overexpression ZC3H13 genes ( B and C ). The expression levels of CD27, CD70, CD80, CD86, ICOS, CTLA4, and LAG3 in siRNA WTAP and overexpression ZC3H13 were demonstrated in Fig. 9D and F, respectively. The result were determined by qRT-PCR. The data are mean ± standard deviation of six replicate experiments. * P < 0.05, **P < 0.01 and ***P < 0.001 compared with the corresponding control values.

Article Snippet: After blocking with 5% nonfat milk, the membranes were immunoblotted with the primary antibodies: WTAP (A04296-2, Boster Biotech, Wuhan, China), CD27 (A01148-2, Boster Biotech), CD70 (A02853-2, Boster Biotech), CD80 (A00196-3, Boster Biotech), CD86(BM4121, Boster Biotech), ICOS (A00291-2, Boster Biotech), CTLA4 (A00020-1, Boster Biotech), LAG3 (M02869-2, Boster Biotech), Beta-actin (BM3873, Boster Biotech).

Techniques: Expressing, Control, Over Expression, Quantitative RT-PCR, Standard Deviation

Journal: Scientific Reports

Article Title: Identification of m6A methyltransferase-related WTAP and ZC3H13 predicts immune infiltrates in glioblastoma

doi: 10.1038/s41598-025-88671-4

Figure Lengend Snippet: Relative expression of genes expression in GBM tissues. Expression levels of WTAP and ZC3H13 genes were compared with those of the control group on GBM tissues, 10 GBM and adjacent tissues, respectively ( A ). The expression levels of siRNA WTAP and overexpression ZC3H13 genes ( B and C ). The expression levels of CD27, CD70, CD80, CD86, ICOS, CTLA4, and LAG3 in siRNA WTAP and overexpression ZC3H13 were demonstrated in Fig. 9D and F, respectively. The result were determined by qRT-PCR. The data are mean ± standard deviation of six replicate experiments. * P < 0.05, **P < 0.01 and ***P < 0.001 compared with the corresponding control values.

Article Snippet: After blocking with 5% nonfat milk, the membranes were immunoblotted with the primary antibodies: WTAP (A04296-2, Boster Biotech, Wuhan, China), CD27 (A01148-2, Boster Biotech), CD70 (A02853-2, Boster Biotech), CD80 (A00196-3, Boster Biotech), CD86(BM4121, Boster Biotech), ICOS (A00291-2, Boster Biotech), CTLA4 (A00020-1, Boster Biotech), LAG3 (M02869-2, Boster Biotech), Beta-actin (BM3873, Boster Biotech).

Techniques: Expressing, Control, Over Expression, Quantitative RT-PCR, Standard Deviation