American Society of Hematology

Figure 3

$Figure 3. Activity of Smo inhibitor NVP-LDE225 on MM cell lines. (A,C) MM cells were cultured for 48 hours in the presence of control medium or NVP-LDE225, and viability was measured by MTT assay. (B) Ptch1 and Gli1 immunoblot of protein lysate from MM cell lines treated with NVP-LDE225 (5μM) for 12, 24, and 36 hours and fractionated by electrophoresis and stained with anti-Ptch1 and anti-Gli1 Abs showing down-regulation of Ptch1 and Gli1, suggesting Smo-dependent mechanisms of Hh activity in these MM cell lines. (D) Ptch1 and Gli1 immunoblot of protein lysate from MM cell lines treated with NVP-LDE225 (5μM) for 12, 24, and 36 hours and fractionated by electrophoresis and stained with anti-Ptch1 and anti-Gli1 Abs showing no down-regulation of Hh genes after treatment, suggesting alternative Smo-independent mechanisms leading to Hh activation in MM. (E) U266 were cultured in the presence of control medium or NVP-LDE225 (5μM) for 24 hours. Immunocytochemical analysis was assessed using anti-Gli1 Ab, and 4,6-diamidino-2-phenylindole was used to stain nuclei. The cells were analyzed using an epifluorescence microscope. Top and middle panels: original magnification ×40. Bottom panel: original magnification ×100. NVP-LDE225 modulates Gli1 activity by inhibiting its nuclear localization. (F) Primary MM cells were cultured for 48 hours in the presence of control medium or NVP-LDE225 in a range of doses from 1 to 20μM (left panel). PBMCs from 5 healthy donors were treated with 20μM NVP-LDE225 up to 72 hours (right panel). Cell viability was evaluated by MTT assay.$

Activity of Smo inhibitor NVP-LDE225 on MM cell lines. (A,C) MM cells were cultured for 48 hours in the presence of control medium or NVP-LDE225, and viability was measured by MTT assay. (B) Ptch1 and Gli1 immunoblot of protein lysate from MM cell lines treated with NVP-LDE225 (5μM) for 12, 24, and 36 hours and fractionated by electrophoresis and stained with anti-Ptch1 and anti-Gli1 Abs showing down-regulation of Ptch1 and Gli1, suggesting Smo-dependent mechanisms of Hh activity in these MM cell lines. (D) Ptch1 and Gli1 immunoblot of protein lysate from MM cell lines treated with NVP-LDE225 (5μM) for 12, 24, and 36 hours and fractionated by electrophoresis and stained with anti-Ptch1 and anti-Gli1 Abs showing no down-regulation of Hh genes after treatment, suggesting alternative Smo-independent mechanisms leading to Hh activation in MM. (E) U266 were cultured in the presence of control medium or NVP-LDE225 (5μM) for 24 hours. Immunocytochemical analysis was assessed using anti-Gli1 Ab, and 4,6-diamidino-2-phenylindole was used to stain nuclei. The cells were analyzed using an epifluorescence microscope. Top and middle panels: original magnification ×40. Bottom panel: original magnification ×100. NVP-LDE225 modulates Gli1 activity by inhibiting its nuclear localization. (F) Primary MM cells were cultured for 48 hours in the presence of control medium or NVP-LDE225 in a range of doses from 1 to 20μM (left panel). PBMCs from 5 healthy donors were treated with 20μM NVP-LDE225 up to 72 hours (right panel). Cell viability was evaluated by MTT assay.

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