Journal: Cell Death & Disease

Article Title: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

doi: 10.1038/s41419-018-0956-4

Figure Lengend Snippet: a CSF1 expression with Hoechst 33342 and their merges of testicular sections from mice under four different treatments in the short term (W2, 2 weeks) and long term (W8, 8 weeks) experiments. Scale bar, 200 μm. b ELISA of CSF1 in mice serum of the four different groups in W2 and W8 experiments. c RT-qPCR detected Csf1 expression in mice testicular tissue under the four different treatments in W2 and W8 experiments. d CSF1 expression level detected by western blot analysis in the four groups in W2 and W8. Histograms are the statistical result of Image J (V1.48d) gradation analysis for the western blot experiments. (The results are shown as the mean ± S.E.M of at least three mice ( n = 3) and the statistical significance was expressed as follows: * p < 0.05; ** p < 0.01). C control group, M normal group treated with MLT, D diabetic group, DM MLT-treated diabetic group

Article Snippet: Mouse CSF1 level of mouse plasma was quantified using an ELISA kit (Boster, Wuhan, China) according to the instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

doi: 10.1038/s41419-018-0956-4

Figure Lengend Snippet: a CSF1 expression with Hoechst 33342 and their merges of MLTC-1 under three different ERS states (C control, Tm Tm treatment induced excessive ERS, Tm + 4PBA Tm treatment in combination with 4PBA for alleviated ERS). b CSF1 concentration in culture medium of MLTC-1 under the three different treatments was detected using ELISA. c RT-qPCR detected the mRNA expression level of Csf1 in MLTC-1 under the three treatments. d Expression of CSF1 in MLTC-1 under different treatments was measured by Western blot. Histograms are the statistical result of Image J (V1.48d) gradation analysis for the western blot experiments. e A graphical conclusion of this study is displayed: DM-induced high glucose activated ERS response factors (Grp78 and CHOP) in Leydig cells, resulting in apoptosis of Leydig cells and growth arrest of SSCs in testes. In response to these changes, melatonin treatment alleviated the apoptosis of Leydig cells via inhibition of ERS and recovered the CSF1 secretion in testes. CSF1 then resumed the self-renewal capacity of SSCs. (The results are expressed as the mean ± S.E.M of three separated wells of cells ( n = 3) in at least three different experiments and the statistical significance is expressed as follows: * p < 0.05; ** p < 0.01)

Article Snippet: Mouse CSF1 level of mouse plasma was quantified using an ELISA kit (Boster, Wuhan, China) according to the instructions.

Techniques: Expressing, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Inhibition

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in squamous cell carcinomas caused by same initial oncogenic drivers

doi: 10.1186/s13046-022-02337-x

Figure Lengend Snippet: TAb2 tumors promote drastic expansion of F4/80 + TAMs. An in vitro co-culture assay was set up using BM cells and TAb2 or TCh3 tumor cells, for evaluating the effects of tumors on myeloid cells. Total numbers of TAMs (CD11b + Ly6C − Ly6G − F4/80 + ) were counted by flow cytometry at different time points (day 2, 3 and 4). A TAb2 tumors drive the expansion of TAMs. BM cells were either cultured alone (BM) or co-cultured with TAb2 (TAb2-BM) or TCh3 (TCh3-BM) tumor cells, respectively. Left panel: Growth curves of F4/80 + TAMs. Right panel: Representative flow plots of CD11b + F4/80 + population. B TAb2 tumors drive the expansion of TAMs independent of cell-cell contact. BM cells were either cultured alone or cultured with TAb2 or TCh3 tumor cells, respectively, in transwell plates. Left panel: Growth curve of F4/80 + TAMs. Right panel: Representative flow plots of CD11b + F4/80 + population. P values are shown for multiple comparisons to TAb2-BM by two-way ANOVA in ( A ) and ( B ). C-D TAb2 tumor-mediated TAM expansion requires CSF1 and VEGF. C Representative flow plots of CD11b vs F4/80 (top) and CD86 vs. CD206 (bottom) in the co-culture of TAb2 tumor cells and BM cells in the absence or presence of CSF1R mAb or VEGFR inhibitor. D Growth curves of F4/80 + TAMs (left) and CD206 + CD86 − M2 TAMs (right). BM cells were co-cultured with TAb2 tumor cells in the absence (black) or presence of CSF1R mAb (red) or VEGFR inhibitor (blue). Results are representative of more than three independent experiments done in triplicates. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test

Article Snippet: For ELISA, cell lysate or supernatant samples were analyzed for cytokine/chemokine levels using the Mouse HGF ELISA Kit (Raybiotech, ELM-HGF), Mouse CXCL17/VCC-1 ELISA Kit (Raybiotech, ELM-CXCL17), Mouse CXCL16 (Sigma-Aldrich, RAB0127), Mouse CXCL12/SDF-1α (Sigma-Aldrich, RAB0125, and Raybiotech, ELM-SDF1a), and Mouse CSF1 (Raybiotech, ELM-MCSF-1) according to the manufacturer’s instructions.

Techniques: In Vitro, Co-culture Assay, Flow Cytometry, Cell Culture, Co-Culture Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in squamous cell carcinomas caused by same initial oncogenic drivers

doi: 10.1186/s13046-022-02337-x

Figure Lengend Snippet: Validation assays show that TAb2 and TCh3 tumors upregulated distinct factors. A Representative images of cytokine array analysis of supernatants of TCh3 (left) and TAb2 (right). Red boxes indicate the cytokines that were visually different between TCh3 and TAb2 tumors. B Quantification of signal differences in cytokine array analysis indicated by the red boxes in ( A ). Left panel: bar graph showing the signal differences between TCh3 and TAb2 tumors. Cytokines shown vertically on the y-axis and signal intensity shown horizontally on the x -axis (positive values for upregulation in TAb2 and negative values for upregulation in TCh3, respectively). Right panel: volcano plot showing adj. p -value of the differences on the y-axis and signal intensity shown horizontally on the x -axis. C Validation of cytokine expression by ELISA. Expression of CXCL16, CXCL17, CSF1 (a.k.a. MCSF), HGF, and CXCL12 from TAb2 or TCh3 cell lysate or supernatant were detected by ELISA. The optical density (OD) at 450 nm is shown on the y-axis and the dilution factor (5-fold serial dilution) is shown on the x-axis. Results are representative of experiments done in duplicates. D TAb2 tumors increased the expression of phosphorylated STAT3 (p-STAT3). Western blotting analysis of total STAT3 and p-STAT3 (Tyr705) in TAb2 and TCh3 cell lysates. GAPDH was used as loading control. Data are representative of at least three independent experiments. E Kaplan-Meier plots of 10-year survival in PIK3CA Amp TP53 Mutated HNSCC patients ( n = 300) expressing different levels of VEGF-C or both CSF1 and VEGF-C. Patients were grouped into high-expression group or low-expression group based on gene expression as described in

Article Snippet: For ELISA, cell lysate or supernatant samples were analyzed for cytokine/chemokine levels using the Mouse HGF ELISA Kit (Raybiotech, ELM-HGF), Mouse CXCL17/VCC-1 ELISA Kit (Raybiotech, ELM-CXCL17), Mouse CXCL16 (Sigma-Aldrich, RAB0127), Mouse CXCL12/SDF-1α (Sigma-Aldrich, RAB0125, and Raybiotech, ELM-SDF1a), and Mouse CSF1 (Raybiotech, ELM-MCSF-1) according to the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Serial Dilution, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in squamous cell carcinomas caused by same initial oncogenic drivers

doi: 10.1186/s13046-022-02337-x

Figure Lengend Snippet: Validation assays show that TAb2 and TCh3 tumors upregulated distinct factors. A Representative images of cytokine array analysis of supernatants of TCh3 (left) and TAb2 (right). Red boxes indicate the cytokines that were visually different between TCh3 and TAb2 tumors. B Quantification of signal differences in cytokine array analysis indicated by the red boxes in ( A ). Left panel: bar graph showing the signal differences between TCh3 and TAb2 tumors. Cytokines shown vertically on the y-axis and signal intensity shown horizontally on the x -axis (positive values for upregulation in TAb2 and negative values for upregulation in TCh3, respectively). Right panel: volcano plot showing adj. p -value of the differences on the y-axis and signal intensity shown horizontally on the x -axis. C Validation of cytokine expression by ELISA. Expression of CXCL16, CXCL17, CSF1 (a.k.a. MCSF), HGF, and CXCL12 from TAb2 or TCh3 cell lysate or supernatant were detected by ELISA. The optical density (OD) at 450 nm is shown on the y-axis and the dilution factor (5-fold serial dilution) is shown on the x-axis. Results are representative of experiments done in duplicates. D TAb2 tumors increased the expression of phosphorylated STAT3 (p-STAT3). Western blotting analysis of total STAT3 and p-STAT3 (Tyr705) in TAb2 and TCh3 cell lysates. GAPDH was used as loading control. Data are representative of at least three independent experiments. E Kaplan-Meier plots of 10-year survival in PIK3CA Amp TP53 Mutated HNSCC patients ( n = 300) expressing different levels of VEGF-C or both CSF1 and VEGF-C. Patients were grouped into high-expression group or low-expression group based on gene expression as described in

Article Snippet: For ELISA, cell lysate or supernatant samples were analyzed for cytokine/chemokine levels using the Mouse HGF ELISA Kit (Raybiotech, ELM-HGF), Mouse CXCL17/VCC-1 ELISA Kit (Raybiotech, ELM-CXCL17), Mouse CXCL16 (Sigma-Aldrich, RAB0127), Mouse CXCL12/SDF-1α (Sigma-Aldrich, RAB0125, and Raybiotech, ELM-SDF1a), and Mouse CSF1 (Raybiotech, ELM-MCSF-1) according to the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Serial Dilution, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in squamous cell carcinomas caused by same initial oncogenic drivers

doi: 10.1186/s13046-022-02337-x

Figure Lengend Snippet: Validation assays show that TAb2 and TCh3 tumors upregulated distinct factors. A Representative images of cytokine array analysis of supernatants of TCh3 (left) and TAb2 (right). Red boxes indicate the cytokines that were visually different between TCh3 and TAb2 tumors. B Quantification of signal differences in cytokine array analysis indicated by the red boxes in ( A ). Left panel: bar graph showing the signal differences between TCh3 and TAb2 tumors. Cytokines shown vertically on the y-axis and signal intensity shown horizontally on the x -axis (positive values for upregulation in TAb2 and negative values for upregulation in TCh3, respectively). Right panel: volcano plot showing adj. p -value of the differences on the y-axis and signal intensity shown horizontally on the x -axis. C Validation of cytokine expression by ELISA. Expression of CXCL16, CXCL17, CSF1 (a.k.a. MCSF), HGF, and CXCL12 from TAb2 or TCh3 cell lysate or supernatant were detected by ELISA. The optical density (OD) at 450 nm is shown on the y-axis and the dilution factor (5-fold serial dilution) is shown on the x-axis. Results are representative of experiments done in duplicates. D TAb2 tumors increased the expression of phosphorylated STAT3 (p-STAT3). Western blotting analysis of total STAT3 and p-STAT3 (Tyr705) in TAb2 and TCh3 cell lysates. GAPDH was used as loading control. Data are representative of at least three independent experiments. E Kaplan-Meier plots of 10-year survival in PIK3CA Amp TP53 Mutated HNSCC patients ( n = 300) expressing different levels of VEGF-C or both CSF1 and VEGF-C. Patients were grouped into high-expression group or low-expression group based on gene expression as described in

Article Snippet: For ELISA, cell lysate or supernatant samples were analyzed for cytokine/chemokine levels using the Mouse HGF ELISA Kit (Raybiotech, ELM-HGF), Mouse CXCL17/VCC-1 ELISA Kit (Raybiotech, ELM-CXCL17), Mouse CXCL16 (Sigma-Aldrich, RAB0127), Mouse CXCL12/SDF-1α (Sigma-Aldrich, RAB0125, and Raybiotech, ELM-SDF1a), and Mouse CSF1 (Raybiotech, ELM-MCSF-1) according to the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Serial Dilution, Western Blot