![Figure 1. Loss of Bim is associated with adaptive bortezomib resistance in Bimhi MM cells. (A) Immunoblotting analysis was performed to profile basal levels of the BH3-only proteins Bim (including EL, L, and S isoforms), Noxa, and/or Puma in untreated human MM cell lines. *Indicates nonspecific bands. (B) Three primary samples obtained from 2 patients with MM as well as normal CD34+ cells isolated from cord blood (CB) were subjected to immunoblotting analysis for basal levels of Bim. Blots for BimEL were quantified by using ImageJ software (available online). Values indicate fold-increase after normalization to loading controls (eg, glyceraldehyde-3-phosphate dehydrogenase [GAPDH] or β-actin [β-act]). (C) Bimhi U266 cells were stably transfected with constructs encoding shRNA-targeting Bim (shBim) or scrambled sequence as a negative control (shNC). Cells were then exposed to 5 nM bortezomib (Btz) for 24 hours, followed by immunoblotting for expression of Bim and cleavage of PARP (left panels) or flow cytometry to determine the percentage of apoptotic (annexin V+) cells (right panel). (D) U266 cells were continuously cultured with gradually increasing concentrations of bortezomib up to 15 nM to generate a subline (PS-R) that acquired marked resistance to bortezomib, as determined by flow cytometry (left panel). A Human Apoptosis RT2 Profiler Array Kit was then used to compare differences in the expression (messenger RNA [mRNA]) of key genes involved in apoptosis between PS-R and U266 cells. The heat map showed genes that were up- or downregulated in PS-R cells compared with U266 cells (right panel). (E) In parallel, immunoblotting analysis was conducted to monitor protein levels of Bim and/or Mcl-1 in untreated parental U266 cells and their bortezomib-resistant counterparts (PS-R). (F) Primary CD138+ cells were isolated from newly diagnosed (Dx; n = 2) or relapsed (R; n = 3, who had received prior bortezomib) MM patients, after which untreated cells were subjected to immunoblotting analysis (left) or immunofluorescence staining for CD138 (phycoerythrin, red) or Bim (AlexaFluor 488, green). Arrows and arrowheads indicate CD138+ cells displaying low and high Bim positivity, respectively. Scale bar = 10 μm; magnification ×40. α-tub, alpha tubulin; CF, cleaved fragment; DAPI, 4′,6 diamidino-2-phenylindole; L.E., long exposure, Veh, vehicle.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/17/10.1182_blood-2014-03-564534/4/2687f1.jpeg?Expires=1735818232&Signature=SVzWKYW0KKwP1HM5vrxWjqLSvwrjP2fGxXA8FGNSCCUUS6lTc~Q40ONRX4M2ZFRg-HMaLJPyuOzpnhe0828Q80yB3ruugAjlpsB74vV7N1~3~HhesfGCEIkecxa3zG-UxpWhyS-9dw85h7x3faflbnd11KJrbmCRxrNsHOVZXEitMnoCyCY~BzVfBm5wZ2liRonP89oGwYyxver5iGMOY9z5RNa9DBKi5ltCn83qUwycvDRncgr1ir3Vrc~zJpoS5FQVARLscj4sU674W1VeyNtya5UAbLknvpRAoH1eJ9F9hJS64YwOfo~DcqaM6lWFthQ9v7DvwjQyFl9alvDPXA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Loss of Bim is associated with adaptive bortezomib resistance in Bimhi MM cells. (A) Immunoblotting analysis was performed to profile basal levels of the BH3-only proteins Bim (including EL, L, and S isoforms), Noxa, and/or Puma in untreated human MM cell lines. *Indicates nonspecific bands. (B) Three primary samples obtained from 2 patients with MM as well as normal CD34+ cells isolated from cord blood (CB) were subjected to immunoblotting analysis for basal levels of Bim. Blots for BimEL were quantified by using ImageJ software (available online). Values indicate fold-increase after normalization to loading controls (eg, glyceraldehyde-3-phosphate dehydrogenase [GAPDH] or β-actin [β-act]). (C) Bimhi U266 cells were stably transfected with constructs encoding shRNA-targeting Bim (shBim) or scrambled sequence as a negative control (shNC). Cells were then exposed to 5 nM bortezomib (Btz) for 24 hours, followed by immunoblotting for expression of Bim and cleavage of PARP (left panels) or flow cytometry to determine the percentage of apoptotic (annexin V+) cells (right panel). (D) U266 cells were continuously cultured with gradually increasing concentrations of bortezomib up to 15 nM to generate a subline (PS-R) that acquired marked resistance to bortezomib, as determined by flow cytometry (left panel). A Human Apoptosis RT2 Profiler Array Kit was then used to compare differences in the expression (messenger RNA [mRNA]) of key genes involved in apoptosis between PS-R and U266 cells. The heat map showed genes that were up- or downregulated in PS-R cells compared with U266 cells (right panel). (E) In parallel, immunoblotting analysis was conducted to monitor protein levels of Bim and/or Mcl-1 in untreated parental U266 cells and their bortezomib-resistant counterparts (PS-R). (F) Primary CD138+ cells were isolated from newly diagnosed (Dx; n = 2) or relapsed (R; n = 3, who had received prior bortezomib) MM patients, after which untreated cells were subjected to immunoblotting analysis (left) or immunofluorescence staining for CD138 (phycoerythrin, red) or Bim (AlexaFluor 488, green). Arrows and arrowheads indicate CD138+ cells displaying low and high Bim positivity, respectively. Scale bar = 10 μm; magnification ×40. α-tub, alpha tubulin; CF, cleaved fragment; DAPI, 4′,6 diamidino-2-phenylindole; L.E., long exposure, Veh, vehicle.
