Figure S3.
Combined loss of SETD1B and KMT2D leads to a more aggressive disease. (A) Illustration of FL adoptive transfer model based on the VavP-BCL2 transgenic and retroviral transduction of HPCs followed by reconstitution in lethally irradiated, syngeneic, female mice. (B) Survival curves for mice harboring sgRNA targeting LacZ (sgLacZ) and mice carrying sgRNA targeting Setd1b (sgSetd1b) (***P = 0.005). P value was calculated using Log-rank (Mantel–Cox) test. (C) Detection of genomic editing of Setd1b sgRNAs by T7 endonuclease I (T7E1) assay. The target fragments were amplified by PCR from genomic DNA from mouse tumors. Arrows indicate the digested fragments by T7EI. Data representative of at least two independent experiments are shown. (D) Representative spleens taken from each mouse group. Spleens from VavP-Bcl2; sgSetd1b (n = 27), VavP-Bcl2; sgKmt2d (n = 8), and VavP-Bcl2; sgkmt2d+sgSetd1b (n = 6) were enlarged compared with the control group, VavP-Bcl2; sgLacZ (n = 22). (E and F) Dot plot graph showing the percentage of positive transduced VavP-Bcl2 HSCs (prior to injection into mice) and splenic lymphoma cells (after injection and disease), indicating cells harboring (E) sgSetd1b-RFP (sgSetd1bRFP) or (F) sgKmt2d-GFP (sgKmt2dGFP) plasmids, (n = 8). (G) Stacked bar graph showing RFP and GFP-positive transduced VavP-Bcl2 HSCs (prior to injection into mice) and splenic lymphoma cells (after injection and disease), indicating cells harboring sgSetd1bRFP, sgKmt2dGFP plasmids or both sgSetd1bRFP and sgKmt2dGFP (RFP; GFP) (n = 3). (H) Additional immunohistochemistry serial micrographs of VavP-Bcl2 spleen tissues extracted from recipient mice upon sacrifice. VavP-Bcl2; sgLacZ, VavP-Bcl2; sgSetd1b, VavP-Bcl2; sgKmt2d, and VavP-Bcl2; sgSetd1b+Kmt2d were stained with H&E, B220, Ki67, PNA, TUNEL, and BCL6 of liver tissues extracted from recipient mice upon sacrifice (n = 3 per group). Insets are 3× magnified. Scale bar, 200 μm, 500 μm. (I) Representative histological micrographs of VavP-Bcl2; sgLacZ versus VavP-Bcl2; sgSetd1b, stained with H&E, B220, PNA, and Ki67 (n = 3 per group). Scale bar, 1 mm. (J) DNA gel representing Ig heavy chain variable region (IgVH) rearrangements in B220+ cells isolated from VavP-Bcl2;sgLacZ and VavP-Bcl2;sgSetd1b spleens. Data representative of two independent experiments are shown. (K) Table summarizing the results of SHM analysis in DNA collected from VavP-Bcl2;sgSetd1b to VavP-Bcl2;sgLacZ tumors. (L and M) H&E and B220 staining of kidney (M) and liver (L) tissues extracted from recipient mice upon sacrifice. n = 3 per group. (N) Flow cytometry analysis of the cellular composition of splenic tumor cells from three mice in each genotype, comparing VavP-Bcl2;sgSetd1b to VavP-Bcl2;sgLacZ. Source data are available for this figure: SourceData FS3.

Combined loss of SETD1B and KMT2D leads to a more aggressive disease. (A) Illustration of FL adoptive transfer model based on the VavP-BCL2 transgenic and retroviral transduction of HPCs followed by reconstitution in lethally irradiated, syngeneic, female mice. (B) Survival curves for mice harboring sgRNA targeting LacZ (sgLacZ) and mice carrying sgRNA targeting Setd1b (sgSetd1b) (***P = 0.005). P value was calculated using Log-rank (Mantel–Cox) test. (C) Detection of genomic editing of Setd1b sgRNAs by T7 endonuclease I (T7E1) assay. The target fragments were amplified by PCR from genomic DNA from mouse tumors. Arrows indicate the digested fragments by T7EI. Data representative of at least two independent experiments are shown. (D) Representative spleens taken from each mouse group. Spleens from VavP-Bcl2; sgSetd1b (n = 27), VavP-Bcl2; sgKmt2d (n = 8), and VavP-Bcl2; sgkmt2d+sgSetd1b (n = 6) were enlarged compared with the control group, VavP-Bcl2; sgLacZ (n = 22). (E and F) Dot plot graph showing the percentage of positive transduced VavP-Bcl2 HSCs (prior to injection into mice) and splenic lymphoma cells (after injection and disease), indicating cells harboring (E) sgSetd1b-RFP (sgSetd1bRFP) or (F) sgKmt2d-GFP (sgKmt2dGFP) plasmids, (n = 8). (G) Stacked bar graph showing RFP and GFP-positive transduced VavP-Bcl2 HSCs (prior to injection into mice) and splenic lymphoma cells (after injection and disease), indicating cells harboring sgSetd1bRFP, sgKmt2dGFP plasmids or both sgSetd1bRFP and sgKmt2dGFP (RFP; GFP) (n = 3). (H) Additional immunohistochemistry serial micrographs of VavP-Bcl2 spleen tissues extracted from recipient mice upon sacrifice. VavP-Bcl2; sgLacZ, VavP-Bcl2; sgSetd1b, VavP-Bcl2; sgKmt2d, and VavP-Bcl2; sgSetd1b+Kmt2d were stained with H&E, B220, Ki67, PNA, TUNEL, and BCL6 of liver tissues extracted from recipient mice upon sacrifice (n = 3 per group). Insets are 3× magnified. Scale bar, 200 μm, 500 μm. (I) Representative histological micrographs of VavP-Bcl2; sgLacZ versus VavP-Bcl2; sgSetd1b, stained with H&E, B220, PNA, and Ki67 (n = 3 per group). Scale bar, 1 mm. (J) DNA gel representing Ig heavy chain variable region (IgVH) rearrangements in B220+ cells isolated from VavP-Bcl2;sgLacZ and VavP-Bcl2;sgSetd1b spleens. Data representative of two independent experiments are shown. (K) Table summarizing the results of SHM analysis in DNA collected from VavP-Bcl2;sgSetd1b to VavP-Bcl2;sgLacZ tumors. (L and M) H&E and B220 staining of kidney (M) and liver (L) tissues extracted from recipient mice upon sacrifice. n = 3 per group. (N) Flow cytometry analysis of the cellular composition of splenic tumor cells from three mice in each genotype, comparing VavP-Bcl2;sgSetd1b to VavP-Bcl2;sgLacZ. Source data are available for this figure: SourceData FS3.

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