HL cells were transfected with this construct by using the CLB Kit (Biozym, Hessisch Oldendorf, Germany) and stable transfectants were determined by treatment with 3 g/mL blasticidin. HL cells were treated for 4 days at a cell density of Mdivi-1 1106 cells/mL with 2.510?4 M all-retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). roscovitin level of sensitivity of HDML-2 cells. Cells of the HL cell collection HDLM-2 were incubated for 5 days with 5-azacytidine or medium. Thereafter, cells were treated with roscovitin. The viability was assessed by propidium iodide staining. The number of living cells in the samples without medicines was arranged as 100%. Offered are means and standard errors from three experiments.(TIF) pone.0055897.s003.tif (262K) GUID:?E18D179C-FC68-4582-8B5F-14197BC04139 Number S4: L-428 cells express PRAME and are resistant against retinoic acid. A) Manifestation of PRAME in the indicated cell lines was assessed by quantitative RT-PCR. For comparative analysis, manifestation in cell collection L-428 was collection as one. ACTB was used as housekeeping control. B) Level of sensitivity of indicated cell lines for RA was assessed by XTT assay. Offered are means and standard errors from five experiments. Asterisks show significance (p<0.05; College students t test).(TIF) pone.0055897.s004.tif (641K) GUID:?14C667B3-23A4-43E4-BDB9-69F4811B6F31 Number S5: L-428 cells express PRAME and are resistant against retinoic acid. Cells of the HL cell collection L-428 with bare vector control (remaining) or L-428 cells after knock-down of PRAME (right) were incubated with 2.510?4 M ATRA IL6 or DMSO. Viability was assessed in emGFP positive cells by propidium iodide staining.(TIF) pone.0055897.s005.tif (19M) GUID:?D33E9941-80DD-43F6-ADC9-6E0750C5D7E8 Figure S6: Knock-down of PRAME increases sensitivity for etoposide. Cells of the HL cell collection L-428 after PRAME knock-down or transfection with control vector were treated for 24 hours with 25 g/mL etoposide. The viability was assessed by propidium iodide staining. The number of living cells in the samples without etoposide was arranged as 100%. Offered are means and standard errors from six experiments. Asterisks show significance (p<0.05; College students t test).(TIF) pone.0055897.s006.tif (2.6M) GUID:?262460A2-E739-4458-84A7-C689C5301608 Figure S7: Expression of TRAIL and additional potential targets after knock-down of PRAME. Cells of the HL cell collection L-428 after PRAME knock-down or transfection with control vector were analyzed by quantitative RT-PCR (top panel). In Mdivi-1 addition, cells were incubated with 2.510?4 M ATRA or DMSO and expression of the indicated gene were again tested by qRT-PCR (lower panel). For comparative analysis expression in control cells without RA were set as one and ACTB was used as housekeeping control. Offered are means and standard errors from three experiments.(TIF) pone.0055897.s007.tif (11M) GUID:?A508375A-6A4F-4568-87C0-8FA0C66FA59A Number S8: HDAC inhibition and PRAME knock-down in L-428 cells. Cells of the HL cell collection L-428 after PRAME knock-down or transfection with control vector were treated for 24 hours with 25 g/mL cisplatin. Cells were pre-incubated with vorinostat or DMSO. The viability was assessed by propidium iodide staining. The number of living cells in the control cells with DMSO without cisplatin was arranged as 100%.(TIF) pone.0055897.s008.tif (2.1M) GUID:?96F6060E-188C-40E2-842E-1A8F6BC04A6A Abstract The prognosis for individuals with Hodgkin lymphoma (HL) has improved in recent decades. On the other hand, not all individuals can be cured with the currently founded therapy regimes and this therapy is associated with several adverse late effects. Therefore it Mdivi-1 is necessary to develop fresh therapy strategies. After treatment of L-540 HL cells with 5-azacytidine (5AC), we observed increased expression of the preferentially indicated antigen in melanoma (PRAME). In addition, we detected an increased resistance of 5AC-treated cells against cytotoxic medicines. We analyzed the influence of PRAME on cell survival of HL cells by knocking down PRAME in the chemotherapy resistant cell collection L-428, a cell collection that communicate PRAME at a high level. After knock-down of PRAME using vector centered RNA interference we observed improved level of sensitivity for cisplatin, etoposide and retinoic acid. DNA microarray analysis of HL cells after PRAME knock-down indicated rules of several genes including down-regulation of known anti-apoptotic factors. Increased retinoic acid signaling in these cells was exposed by increased manifestation of the retinoic acid metabolizing cytochrome P450 (CYP26B1), a transcriptional target of retinoic Mdivi-1 acid signaling. Our data suggest that PRAME inhibits retinoic acid.