Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a-c , CD8 + T cells were treated with EPZ004777 for 48 hours. Western Blot showed H3K79me2 in CD8 + T cells ( a ). FACS demonstrated CD8 + T cell apoptosis ( b ) and cytokines ( c ). d , Western Blot showed H3K79me2 in Dot1l f/f and Dot1l −/− CD8 + T cells. e, f , Effect of DOT1L deletion on CD8 + T cells apoptosis ( e ) and cytokines ( f ). g-i , Effect of T cell DOT1L deficiency on MC38 growth ( g, h ) and T cell viability ( i ). j, k , Effect of methionine supplementation on Dot1l f/f and Dot1l −/− CD8 + T cells apoptosis ( j ) and cytokines ( k ). l , GSEA plot showed enriched apoptotic pathway in Dot1l −/− CD8 + T cells. m , Heat map showed Jak-Stats mRNA levels in mouse Dot1l −/− vs Dot1l f/f CD8 + T cells. n , Western blot showed STAT5 and p-STAT5 in Dot1l f/f ( f/f ) and Dot1l −/− (−/−) CD8 + T cells. o , Western blot showed STAT5 and p-STAT5 in CD8 + T cells cultured with fresh medium (FM), Supernatant (Sup), and Sup supplemented with different metabolites (Sup+). p , ChIP assay showed H3K79me2 occupancy on the Stat5b promoter in CD8 + T cells. q , ChIP assay showed H3K79me2 occupancy on the Stat5b promoter in CD8 + T cells cultured with FM, Sup, or Sup+Met. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Western Blot, Cell Culture

Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a, b , H3K79me2 ( a ) and STAT5 ( b ) levels in CD8 + T cells from tumor draining lymph node (dLN) and tumor in B16F10 bearing mice. c, d , H3K79me2 ( c ) and STAT5 ( d ) levels in CD8 + T cells from spleen and tumor ascites in ID8 bearing mice. e , H3K79me2 levels in CD8 + T cells from healthy peripheral blood and human ovarian cancers ascites. f, g , H3K79me2 ( f ) and STAT5 ( g ) levels in CD8 + T cells from healthy human blood and human ovarian cancer omentum tissues. h, i , FACS showed H3K79me2 and STAT5 levels in human tumor infiltrating CD8 + T cells. j-m , Effect of methionine on human tumor infiltrating CD8 + T cells. Human colorectal cancer infiltrating CD8 + T cells were cultured with or without methionine. T cell cytokine production ( j, k ), H3K79me2 ( l ), and STAT5 ( m ) were analyzed by FACS. One representative of four is shown. n , Effect of methionine supplementation on apoptosis of tumor infiltrating CD8 + T cells and ID8 tumor cells in vivo. ID8 tumor bearing mice were treated with methionine or PBS. T cell and tumor cell apoptosis was determined by FACS. o , Methionine levels in ID8 tumor after methionine or PBS treatment. p-r: Effect of anti-PD-L1 on methionine-affected CT26 tumor progression. Mice bearing CT26 tumor were treated with anti-PD-L1, methionine, and their combination. Tumor volume ( p ), T cell tumor infiltration ( q ) and apoptosis ( r ) were assessed. Data are mean ± s.e.m. Information on sample sizes, experimental number, times, biological replicates, statistical tests, and P values is available in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Cell Culture, In Vivo

Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a-e , Methionine supplementation restored T cell immunity in B16F10-bearing mice. Tumor growth ( a ), tumor infiltrating CD8 + T cell H3K79me2 ( b ) and STAT5 ( c ) were monitored. FACS showed intratumor CD8 + T cell apoptosis ( d ) and cytokines ( e ) . f-h , Methionine supplementation restored T cell immunity in ID8-bearing mice. Tumor growth was monitored by bioluminescence imaging ( f ). FACS showed ascites CD8 + T cell ( g ) and intratumor CD8 + T cell cytokines ( h ) . i-l , Studies on colorectal cancer patients treated with methionine. Western blot showed p-STAT5 and H3K79me2 in peripheral CD8 + T cells prior and post methionine treatment ( i ). FACS showed IL-2 + T cells ( j ), CD8 + T cell effector cytokines ( k ) and apoptosis ( l ) in patients prior and post methionine treatment. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Imaging, Western Blot