rag1 ko mice  (Jackson Laboratory)

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A, B ) Mouse HCC cells, Hepa1-6 or Hep53.4, were subcutaneously injected into Rag1 −/− C57BL/6 or immunocompetent C57BL/6 mice ( n = 5–7 per group). When bore visible tumors, 5 mg/kg citalopram was treated daily for 15 or 25 days. Tumors were excised after mice were sacrificed, and the tumor weight was measured. ( C, D ) Western blotting showed the knockdown efficiency of SERT in Hepa1-6 and Hep53.4 cells. ( E, F ) shControl and sh Slc6a4 Hepa1-6 and Hep53.4 cells were subcutaneously injected into Rag1 −/− C57BL/6 or immunocompetent C57BL/6 mice ( n = 5–6 per group). When bore visible tumors, 5 mg/kg citalopram was treated daily for 15 or 25 days. Tumors were excised after mice were sacrificed, and the tumor weight was measured. In all panels, *p < 0.05, **p < 0.01, ***p < 0.001. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method (for bar chart comparison) and two-way ANOVA with Dunnett’s multiple comparisons (for survival curve comparison). Figure 1—source data 1. Original western blots for , indicating the relevant bands. Figure 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: In the subcutaneous xenograft model, Rag1 −/− C57BL/6 mice (male, 6- to 8-week-old, Cyagen Biosciences, C001197), wild type immune-competent C57BL/6 mice (male or female, 6- to 8-week-old) or Tph1 −/− mice (male, 6- to 8-week-old, Cyagen Biosciences, S-KO-05541), were maintained on a 12-hr day/night cycle with ad libitum access to food and water.

Techniques: Injection, Western Blot, Knockdown, Comparison

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A, B ) Immunohistochemical analysis of cleaved caspase-3 (CCS3) and Ki67 in Hepa1-6-bearing subcutaneous xenograft tumors from Rag1 −/− or immunocompetent C57BL/6 mice, treated with DMSO or 5 mg/kg citalopram. ( C, D ) Immunohistochemical analysis of CCS3 and Ki67 in Hep53.4-bearing subcutaneous xenograft tumors from Rag1 −/− or immunocompetent C57BL/6 mice, treated with DMSO or 5 mg/kg citalopram. In all panels, **p < 0.01, ***p < 0.001. Scale bar, 50 μm. Values are presented as mean ± SD and compared by the Student’s t test.

Article Snippet: In the subcutaneous xenograft model, Rag1 −/− C57BL/6 mice (male, 6- to 8-week-old, Cyagen Biosciences, C001197), wild type immune-competent C57BL/6 mice (male or female, 6- to 8-week-old) or Tph1 −/− mice (male, 6- to 8-week-old, Cyagen Biosciences, S-KO-05541), were maintained on a 12-hr day/night cycle with ad libitum access to food and water.

Techniques: Immunohistochemical staining

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Plate colony formation assay revealed the effect of SERT knockdown alone or combined with citalopram treatment on the long-term cell proliferation of Hepa1-6 and Hep53.4 cells ( n = 6 per group). ( B ) Caspase-3/7 activity in Hepa1-6 and Hep53.4 cells upon SERT knockdown or combined treatment with 5 μM citalopram ( n = 6 per group). ( C, D ) Immunohistochemical analysis of cleaved caspase-3 (CCS3) and Ki67 in shControl and sh Slc6a4 Hepa1-6-bearing subcutaneous xenograft tumors from Rag1 −/− or immunocompetent C57BL/6 mice, treated with DMSO or 5 mg/kg citalopram. Scale bar, 50 μm. ( E, F ) Immunohistochemical analysis of CCS3 and Ki67 in shControl and sh Slc6a4 Hep53.4-bearing subcutaneous xenograft tumors from Rag1 −/− or immunocompetent C57BL/6 mice, treated with DMSO or 5 mg/kg citalopram. ( G ) Single-cell analysis of the expression pattern of SLC6A4 in a HCC cohort ( GSE125449 ). Scale bar, 50 μm. In all panels, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 50 μm. Values as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups.

Article Snippet: In the subcutaneous xenograft model, Rag1 −/− C57BL/6 mice (male, 6- to 8-week-old, Cyagen Biosciences, C001197), wild type immune-competent C57BL/6 mice (male or female, 6- to 8-week-old) or Tph1 −/− mice (male, 6- to 8-week-old, Cyagen Biosciences, S-KO-05541), were maintained on a 12-hr day/night cycle with ad libitum access to food and water.

Techniques: Colony Assay, Knockdown, Activity Assay, Immunohistochemical staining, Single-cell Analysis, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Western blotting showed the knockdown efficiency of GLUT1 in mouse Hepa1-6 cells. ( B ) GLUT1 KD Hepa1-6 cells were subcutaneously injected into the Rag1 −/− or immunocompetent C57BL/6 mice, and mice were treated with 5 mg/kg citalopram when bore visible tumors; 3 weeks later, tumor burden was examined ( n = 6–7 per group). ( C ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host ( n = 7). ( D ) Immunofluorescence analysis of C5a deposition in GLUT1 KD Hepa1-6 tumors from C5ar1 +/− and C5ar1 −/− C57BL/6 host. Scale bar, 50 μm. ( E ) Experimental design of bone marrow transfer experiments. ( F, G, I ) GLUT1 KD Hepa1-6 cells were subcutaneously implanted into syngeneic recipient (r) mice that had been reconstituted with bone marrow cells from either C5ar1 +/− or C5ar1 −/− donor mice. The therapeutic effect of citalopram ( F ), C5a deposition ( G ), and macrophage phagocytosis ( I ) in this model was analyzed. Scale bar, 50 μm. ( H ) The phagocytic capacity of macrophages isolated from GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host. Flow cytometry showed the infiltration of CD45 + CD11b + F4/80 + macrophages ( J ), CD206 + TAMs and CD11b + TAMs ( K ), tumor-infiltrating lymphocytes ( L ) in tumor tissues from orthotopic xenograft model, which was generated in immunocompetent C57BL/6 mice with Hepa1-6 cells ( n = 5 per group). ( M, N ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in C and F . ( O ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host upon CD8 + T cell depletion ( n = 7). ( P ) Correlation analysis of C5aR1 expression and immune checkpoint molecules, gene signatures of TAMs, exhausted T cells, and effector Tregs in the TCGA cohort ( n = 371). In all panels, *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. Values are presented as mean ± SD and compared by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons ( B, C, F, O ), Student’s t test ( H–M ), one-way ANOVA multiple comparisons with Tukey’s method ( B, N ), and the Spearman’s rank correlation methods ( P ). Figure 3—source data 1. Original western blots for , indicating the relevant bands. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: In the subcutaneous xenograft model, Rag1 −/− C57BL/6 mice (male, 6- to 8-week-old, Cyagen Biosciences, C001197), wild type immune-competent C57BL/6 mice (male or female, 6- to 8-week-old) or Tph1 −/− mice (male, 6- to 8-week-old, Cyagen Biosciences, S-KO-05541), were maintained on a 12-hr day/night cycle with ad libitum access to food and water.

Techniques: Western Blot, Knockdown, Injection, Immunofluorescence, Isolation, Flow Cytometry, Generated, Cell Function Assay, Expressing