Figure 1
Figure 1. miR-155 expression in hematopoietic cell subsets of normal and tumor-bearing mice. (A) Experimental scheme: The BM of 5.5-week-old PyMT mice was reconstituted with wild-type FVB HSPCs transduced with miR-155 sensor (BdLV.155T) or Ctrl (BdLV.Ctrl) vectors. The chronology of breast cancer development and progression is indicated. (B) Representative dot plots show reporter expression (y-axis: GFP, miRNA reporter; x-axis: NGFR, normalizer) in tumor-infiltrating CD4+ lymphocytes (upper panel) or CD11b+ myeloid cells (lower panel) comparing BdLV.Ctrl (left) with BdLV.155T (right). Quantification of miR-155 activity in the indicated tumor-infiltrating hematopoietic subpopulations was performed as described in Materials and methods. Shown is the mean “fold repression” of BdLV.155T with respect to BdLV.Ctrl ± SEM, n = 7 to 12 mice. (C) miR-155 activity in splenocytes recovered from 13-week-old PyMT mice is shown, along with the mean fold repression of BdLV.155T with respect to BdLV.Ctrl ± SEM, n = 6 to 10 mice. (D) The graph shows miR-155 activity in splenocytes of wild-type mice with (gray) or without (white) in vivo LPS treatment (median ± range, n = 2). (E) Shown is the scheme of BM-derived dendritic cell (BMDC) differentiation protocol. miR-155 activity in BMDCs treated with LPS for 48 hours (redundant, since we stated in materials and methods the we show the mean +− sem unless otherwise noted n = 3) is shown in the graph (bottom left). qPCR of the indicated miRNAs in BMDCs that were or were not treated with LPS for 12 hours is shown in the graph (bottom right). The data show the relative abundance of each miRNA after normalization to miR-16 (n = 3). (F) Tumor-infiltrating hematopoietic cells were sorted from breast cancers of 13-week-old PyMT mice, as shown (top panels). miR-155 expression was measured in tumor-infiltrating macrophages (Mrc1+ or CD11c+ subsets) and CD4+ T lymphocytes by qPCR (bottom panel). Shown are the 2−ΔCt values using miR-16 as a normalizer (n = 6 from 2 independent experiments). **P < .01; *** P < .001. eGFP, enhanced green fluorescent protein; NGFR, truncated low affinity nerve growth factor receptor; PGK, phosphoglycerate kinase; SA, splice acceptor; SD, splice donor; WPRE, woodchuck hepatitis posttranscriptional regulatory element.

miR-155 expression in hematopoietic cell subsets of normal and tumor-bearing mice. (A) Experimental scheme: The BM of 5.5-week-old PyMT mice was reconstituted with wild-type FVB HSPCs transduced with miR-155 sensor (BdLV.155T) or Ctrl (BdLV.Ctrl) vectors. The chronology of breast cancer development and progression is indicated. (B) Representative dot plots show reporter expression (y-axis: GFP, miRNA reporter; x-axis: NGFR, normalizer) in tumor-infiltrating CD4+ lymphocytes (upper panel) or CD11b+ myeloid cells (lower panel) comparing BdLV.Ctrl (left) with BdLV.155T (right). Quantification of miR-155 activity in the indicated tumor-infiltrating hematopoietic subpopulations was performed as described in Materials and methods. Shown is the mean “fold repression” of BdLV.155T with respect to BdLV.Ctrl ± SEM, n = 7 to 12 mice. (C) miR-155 activity in splenocytes recovered from 13-week-old PyMT mice is shown, along with the mean fold repression of BdLV.155T with respect to BdLV.Ctrl ± SEM, n = 6 to 10 mice. (D) The graph shows miR-155 activity in splenocytes of wild-type mice with (gray) or without (white) in vivo LPS treatment (median ± range, n = 2). (E) Shown is the scheme of BM-derived dendritic cell (BMDC) differentiation protocol. miR-155 activity in BMDCs treated with LPS for 48 hours (redundant, since we stated in materials and methods the we show the mean +− sem unless otherwise noted n = 3) is shown in the graph (bottom left). qPCR of the indicated miRNAs in BMDCs that were or were not treated with LPS for 12 hours is shown in the graph (bottom right). The data show the relative abundance of each miRNA after normalization to miR-16 (n = 3). (F) Tumor-infiltrating hematopoietic cells were sorted from breast cancers of 13-week-old PyMT mice, as shown (top panels). miR-155 expression was measured in tumor-infiltrating macrophages (Mrc1+ or CD11c+ subsets) and CD4+ T lymphocytes by qPCR (bottom panel). Shown are the 2−ΔCt values using miR-16 as a normalizer (n = 6 from 2 independent experiments). **P < .01; *** P < .001. eGFP, enhanced green fluorescent protein; NGFR, truncated low affinity nerve growth factor receptor; PGK, phosphoglycerate kinase; SA, splice acceptor; SD, splice donor; WPRE, woodchuck hepatitis posttranscriptional regulatory element.

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