American Society of Hematology

Figure 3.

$Exposure to dexamethasone (DEX) does not alter FOXO or GSK3β activity in HMCLs. (A) Representative western blot showing FOXO1 and FOXO3a protein expression in nuclear and cytosolic fractions of the indicated HMCLs after 4 hours of exposure to 1 μM DEX, 100 nM copanlisib (COP), or negative control (ctr). α-tubulin and lamin B1 were used as loading controls for cytosolic and nuclear fractions, respectively. (B) Representative image-based flow cytometry image captures by ImageStream at original 60× magnification from indicated HMCLs exposed for 4 hours to DEX 1 μM, COP 100 nM, or negative control. Single images were taken for nuclear dye DR and FOXO (FOXO3a for MM1.s and OPM-2 and FOXO1 for L363) and overlaid with bright-field images. (C) Histograms showing colocalization between DR and FOXO of ImageStream data as obtained in panel B. A positive value indicates similar pixel occupancy, and therefore colocalization, between DR and FOXO, whereas a negative value indicates no similarity in pixel occupancy between both signals. (D) Representative western blot showing expression of GSK3β phosphorylated on serine residue 9 (pGSK3β S9) in indicated HMCLs after 4 hours of exposure to 1 μM DEX, 100 nM COP, or control. α-tubulin was used as a loading control. Quantification findings are given in supplemental Figure 6.$

Exposure to dexamethasone (DEX) does not alter FOXO or GSK3β activity in HMCLs. (A) Representative western blot showing FOXO1 and FOXO3a protein expression in nuclear and cytosolic fractions of the indicated HMCLs after 4 hours of exposure to 1 μM DEX, 100 nM copanlisib (COP), or negative control (ctr). α-tubulin and lamin B1 were used as loading controls for cytosolic and nuclear fractions, respectively. (B) Representative image-based flow cytometry image captures by ImageStream at original 60× magnification from indicated HMCLs exposed for 4 hours to DEX 1 μM, COP 100 nM, or negative control. Single images were taken for nuclear dye DR and FOXO (FOXO3a for MM1.s and OPM-2 and FOXO1 for L363) and overlaid with bright-field images. (C) Histograms showing colocalization between DR and FOXO of ImageStream data as obtained in panel B. A positive value indicates similar pixel occupancy, and therefore colocalization, between DR and FOXO, whereas a negative value indicates no similarity in pixel occupancy between both signals. (D) Representative western blot showing expression of GSK3β phosphorylated on serine residue 9 (pGSK3β S9) in indicated HMCLs after 4 hours of exposure to 1 μM DEX, 100 nM COP, or control. α-tubulin was used as a loading control. Quantification findings are given in supplemental Figure 6.

This Feature Is Available To Subscribers Only