American Society of Hematology

Figure 2

$Figure 2. Expression of Hh components in MM cell lines. (A) MM cell lines were double-stained with anti-CD138–APC and anti-Shh–PE Abs, and the percentage of positive cells was analyzed by a FACSCanto II flow cytometer. Shh is expressed in CD138+ as well CD138− population. (B-D) MM cells were stained with 2 different anti-Ptch1 primary Abs (B) or anti-Smo primary Abs (C-D) followed by specific FITC-conjugated secondary Abs and analyzed with flow cytometry. For both Ptch1 and Smo, similar staining patterns were observed. In both experiments, background staining has been excluded using cells stained with secondary Ab FITC-conjugated as negative control. (C) Lower panel: Smo immunoblot of protein lysate from MM cell lines fractionated by electrophoresis and stained with anti-Smo Ab showing basal level of Smo expression in MM cell lines. (E) MM1S cells were cultured in control medium (top panel) or in the presence of forskolin (FSK; 10μM; bottom panel) for 24 hours. FSK inhibits the nuclear translocation of Gli1. Immunocytochemical analysis was assessed using anti-Gli1Ab, and 4,6-diamidino-2-phenylindole was used to stain nuclei. The cells were analyzed using an epifluorescence microscope (Nikon Eclipse E800, Nikon) and a Photometrics Coolsnap CF color camera (Nikon). (E) Original magnification ×100. Gli1 expression and its nuclear localization suggestive of Hh pathway activity were observed in MM cells. The inhibition by FSK suggests a Smo-independent activation of Gli1.$

Expression of Hh components in MM cell lines. (A) MM cell lines were double-stained with anti-CD138–APC and anti-Shh–PE Abs, and the percentage of positive cells was analyzed by a FACSCanto II flow cytometer. Shh is expressed in CD138⁺ as well CD138⁻ population. (B-D) MM cells were stained with 2 different anti-Ptch1 primary Abs (B) or anti-Smo primary Abs (C-D) followed by specific FITC-conjugated secondary Abs and analyzed with flow cytometry. For both Ptch1 and Smo, similar staining patterns were observed. In both experiments, background staining has been excluded using cells stained with secondary Ab FITC-conjugated as negative control. (C) Lower panel: Smo immunoblot of protein lysate from MM cell lines fractionated by electrophoresis and stained with anti-Smo Ab showing basal level of Smo expression in MM cell lines. (E) MM1S cells were cultured in control medium (top panel) or in the presence of forskolin (FSK; 10μM; bottom panel) for 24 hours. FSK inhibits the nuclear translocation of Gli1. Immunocytochemical analysis was assessed using anti-Gli1Ab, and 4,6-diamidino-2-phenylindole was used to stain nuclei. The cells were analyzed using an epifluorescence microscope (Nikon Eclipse E800, Nikon) and a Photometrics Coolsnap CF color camera (Nikon). (E) Original magnification ×100. Gli1 expression and its nuclear localization suggestive of Hh pathway activity were observed in MM cells. The inhibition by FSK suggests a Smo-independent activation of Gli1.

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