Figure 5
Figure 5. Effect of SPRY2 on B1-cell development, and BCR signaling in murine model and SPRY2 knockdown leads to disease progression in NSG mice. (A) Western blot showing overexpression of spry2 in splenic B cells of CD19-cre;Spry2(tg) mice (left). B1 cells were isolated from the peritoneal cavity of CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice. B1 cells were stained with CD5 and B220 dye to determine the frequency of B1 cells using flow cytometric analysis. Displayed is a dot plot showing B1a-cells frequency in CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice (middle). Bar graph of absolute number of B1a cells in the peritoneal cavity of CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice (right). (B) Splenic B cells were isolated by negative selection from rtTA-positive CD19-cre;Spry2(wt) (top, left) and CD19-cre;Spry2(tg) (top, right) mice. GFP+ population displays CD19-cre–expressed cells and displayed is calcium mobilization assay of splenic B cells from above described mice (bottom). n = 3; P = .0002. (C) Splenic B cells were isolated from CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice, stained with CFSE dye and cultured in vitro for 6 days with and without anti-IgM antibody and LPS. Displayed is the CFSE labeling of splenic B cells of CD19-cre;Spry2(wt) (top) and CD19-cre;Spry2(tg) (bottom) mice. SPRY2 was stably knocked down in Mec-1 CLL cells using pLenti-siRNA A and pLenti-siRNA B, and pLenti-scr was used as control. (D) Average of tumor volume in mice in each group were transplanted with 1.5 million pLenti-scr and pLenti-siRNA B Mec-1 CLL cells (n = 5). Tumor volume was measured using a digital caliper, and we determined the length and width of the tumor. Tumor volume was calculated by V = (L × W × W) / 2 where V is the tumor volume, L is the tumor length, and W is the tumor width. (E) Frequency of human CD19+ cells in spleen of pLenti-scr and pLenti-siRNA B CLL-cell–transplanted mice. (F) Hematoxylin and eosin staining with tissue section of kidney, liver, and spleen to observe the number of CLL-cell infiltration in pLenti-scr and pLenti-siRNA B CLL-cell–transplanted mice. (G) Protein lysates were prepared from tumors; equal amount of protein was loaded in each lane of 10% sodium dodecyl sulfate gel. Shown is scanned western blot to determine the protein levels of p-Erk and SPRY2 in tumors from pLenti-scr and pLenti-siRNA B CLL-cell–transplanted mice. Total Erk and β-actin were used as control. Densitometric measurements showing elevated p-Erk, normalized by total Erk in SPRY2 knockdown tumors. LPS, lipopolysaccharide; rtTA, reverse tetracycline transactivator; WT, wild-type.

Effect of SPRY2 on B1-cell development, and BCR signaling in murine model and SPRY2 knockdown leads to disease progression in NSG mice. (A) Western blot showing overexpression of spry2 in splenic B cells of CD19-cre;Spry2(tg) mice (left). B1 cells were isolated from the peritoneal cavity of CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice. B1 cells were stained with CD5 and B220 dye to determine the frequency of B1 cells using flow cytometric analysis. Displayed is a dot plot showing B1a-cells frequency in CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice (middle). Bar graph of absolute number of B1a cells in the peritoneal cavity of CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice (right). (B) Splenic B cells were isolated by negative selection from rtTA-positive CD19-cre;Spry2(wt) (top, left) and CD19-cre;Spry2(tg) (top, right) mice. GFP+ population displays CD19-cre–expressed cells and displayed is calcium mobilization assay of splenic B cells from above described mice (bottom). n = 3; P = .0002. (C) Splenic B cells were isolated from CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice, stained with CFSE dye and cultured in vitro for 6 days with and without anti-IgM antibody and LPS. Displayed is the CFSE labeling of splenic B cells of CD19-cre;Spry2(wt) (top) and CD19-cre;Spry2(tg) (bottom) mice. SPRY2 was stably knocked down in Mec-1 CLL cells using pLenti-siRNA A and pLenti-siRNA B, and pLenti-scr was used as control. (D) Average of tumor volume in mice in each group were transplanted with 1.5 million pLenti-scr and pLenti-siRNA B Mec-1 CLL cells (n = 5). Tumor volume was measured using a digital caliper, and we determined the length and width of the tumor. Tumor volume was calculated by V = (L × W × W) / 2 where V is the tumor volume, L is the tumor length, and W is the tumor width. (E) Frequency of human CD19+ cells in spleen of pLenti-scr and pLenti-siRNA B CLL-cell–transplanted mice. (F) Hematoxylin and eosin staining with tissue section of kidney, liver, and spleen to observe the number of CLL-cell infiltration in pLenti-scr and pLenti-siRNA B CLL-cell–transplanted mice. (G) Protein lysates were prepared from tumors; equal amount of protein was loaded in each lane of 10% sodium dodecyl sulfate gel. Shown is scanned western blot to determine the protein levels of p-Erk and SPRY2 in tumors from pLenti-scr and pLenti-siRNA B CLL-cell–transplanted mice. Total Erk and β-actin were used as control. Densitometric measurements showing elevated p-Erk, normalized by total Erk in SPRY2 knockdown tumors. LPS, lipopolysaccharide; rtTA, reverse tetracycline transactivator; WT, wild-type.

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