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. 2010 Aug 2;30(19):4575–4594. doi: 10.1128/MCB.01595-09

FIG. 5.

FIG. 5.

ICSBP expression influences calpain activity in undifferentiated myeloid cells. (A) There is less calpain activity in myeloid progenitor cells from ICSBP−/− mice than in WT cells. Myeloid progenitor cells were isolated from the bone marrow of WT or ICSBP−/− mice and cultured in GM-CSF, IL-3, and SCF. ICSBP−/− myeloid progenitors were transduced with retroviral vectors to express Gas2-specific shRNA (or scrambled control shRNA), a dominant-negative (DN) Gas2, or Bcr/abl (or empty vector control). WT progenitor cells were transduced with the Bcr/abl expression vector (or vector control) with or without a vector to express DN-Gas2 (or empty vector control) or a Gas2-specific shRNA (or scrambled control shRNA). Cell lysates were analyzed for calpain activity. *, statistically significant increase in calpain activity with the expression of Gas2-specific shRNA in ICSBP−/− myeloid progenitors (P < 0.001, n = 4); **, statistically significant increase in calpain activity in WT versus ICSBP−/− myeloid progenitors (P < 0.0001, n = 4); ***, statistically significant increase in calpain activity in ICSBP−/− myeloid progenitor cells expressing DN-Gas2 (P < 0.001, n = 4); # or &, statistically significant decrease in calpain activity in Bcr/abl+ WT myeloid progenitor cells (P < 0.001, n = 4). ##, statistically significant increase in calpain activity in WT cells expressing Gas2-specific shRNA (P < 0.001, n = 4); ###, statistically significant decrease in calpain activity in these cells also transduced with Bcr/abl expression vector (P < 0.001, n = 4); && or &&&, statistically significant increase in calpain activity in WT myeloid progenitors expressing DN-Gas2 or decrease in calpain activity in these cells, respectively, upon Bcr/abl expression (P < 0.001, n = 4). The relative expression levels of Gas2 in these cells are indicated by the results in the inset Western blot. (B) Calpain activity in U937 cells is increased by overexpression of ICSBP or Gas2 and decreased by ICSBP knockdown, Bcr/abl, or dominant-negative Gas2. U937 cells were transfected with a dose titration of vectors to overexpress Gas2, dominant-negative Gas2 (DN-Gas2), or a Gas2-specific shRNA (or relevant control vectors). An amount of Gas2, DN-Gas2, or shGas2 expression vector with minimal impact on calpain activity was identified. Based on these results, stable U937 transfectants expressing Bcr/abl (or vector control) or an ICSBP-specific shRNA (or scrambled shRNA) were cotransfected with DN-Gas2 expression vector (or vector control) or shGas2 expression vector (or control). Some Bcr/abl (or control) transfectants were cotransfected with an ICSBP expression vector. Cell lysates were analyzed for calpain activity. *, statistically significant decrease in calpain activity with Gas2 overexpression (P < 0.002, n = 4); ** or #, statistically significant increase in calpain activity with DN-Gas2 or shGas2 expression, respectively (P < 0.001, n = 4); *** or ##, statistically significant decrease in calpain activity with Bcr/abl or ICSBP-specific shRNA expression, respectively (P < 0.0001, n = 5); ###, statistically significant increase in calpain activity in cells with ICSBP-specific shRNA and DN-Gas2 or shGas2 (P < 0.01, n = 4); &, statistically significant increase in calpain activity in Bcr/abl-expressing cells with ICSBP overexpression (P = 0.001, n = 5). (C) Interaction between Gas2 and calpain is decreased by ICSBP overexpression and increased by ICSBP knockdown or Bcr/abl expression in U937 cells. Cell lysates from these U937 transfectants were analyzed for interaction between Gas2 and calpain by coimmunoprecipitation (IP). Cell lysates were immunoprecipitated with antibody to the small subunit of calpain or irrelevant control antibody. Immunoprecipitates were separated by SDS-PAGE, and Western blots (WB) probed with antibody to Gas2. Blots were reprobed with a calpain antibody as a loading control. The bottom panel shows the results for Gas2 abundance (as determined by pixel count) normalized to the abundance of calpain which immunoprecipitated in each experiment (in pixels). No proteins were detectable in control antibody immunoprecipitates, which are not shown. (D) Gas2 protein abundance decreases and calpastatin protein abundance increases during myeloid differentiation, and this influences the interaction of these proteins with calpain. Cell lysates from untreated or IFN-γ-differentiated U937 cells were immunoprecipitated with an antibody to the common small subunit of calpain (or irrelevant control antibody). Immunoprecipitated proteins were separated by SDS-PAGE, and coprecipitating proteins were identified by serially probing Western blots with antibodies to Gas2, calpastatin, and calpain. The input lysate protein (1/10 amount) was a loading control in these studies (blots were probed with antibody to tubulin). (E) The abundance of Gas2 and calpastatin proteins influences the relative inhibition of calpain by these proteins during myeloid differentiation. Lysate proteins from untreated or IFN-γ-differentiated U937 cells were immunoprecipitated with an antibody to Gas2 or calpastatin (or with irrelevant control antibody). Immunoprecipitates were incubated with recombinant calpain, and calpain assays were performed. *, statistically significant difference in calpain activity in experiments with Gas2 from undifferentiated U937 cells versus that in experiments with calpastatin or control studies (P < 0.001, n = 3); ** or ***, statistically significant difference in calpain activity in experiments with calpastatin or Gas2, respectively, from undifferentiated versus differentiated U937 cells (P < 0.001, n = 3). (F) The expression of various forms of Gas2 in U937 cells transfected with a DN-Gas2 expression vector is shown. Lysate proteins from stable U937 transfectants with a vector to express DN-Gas2 or the vector control were separated by SDS-PAGE, and Western blots were serially probed with antibodies to Gas2, caspase 3, caspase 7, and tubulin (as a loading control). In vitro-translated 35S-labeled proteins representing Gas2, Gas2(1-279), and DN-Gas2 were separated on the same SDS-PAGE gel. An autoradiograph of the in vitro-translated (IVT) protein lanes was compared to the Western blot probed for Gas2 to determine the forms of Gas2 present in these cells. Molecular sizes in kDa are shown to the left. Error bars indicate standard errors.