Article Figures & Data
Figures
- Figure 1.
Constitutive and cytokine-mediated regulation of HLA class I and class II surface expression. A, constitutive and cytokine-mediated dose-dependent upregulation of HLA class I surface antigen expression. B, constitutive and cytokine-mediated time-dependent upregulation of HLA class I and HLA class II antigen surface expression. The cells were either left untreated or treated with 200 units of IFN-γ for the time points indicated. Flow cytometry was carried out using FITC-conjugated HLA class I- and class II-specific mAb. Results are expressed as MFI ± SD. The experiments were carried out at least 3 times. C, antiproliferative effect of IFN-γ in Colo 794. Cells were treated with the indicated concentrations of IFN-γ for 48 hours and the number of viable cells was quantified by XTT measurements. Results are expressed as relative growth to untreated cells. D, presence of IFN-γR on the cell surface expression. The IFN-γR expression was determined by flow cytometry by using IFN-γR chain-specific mAbs. The results are presented as histograms. The dotted line represents the IgG1 isotype control, the thin line the IFN-γR1, and the thick line the IFN-γR2.
- Figure 2.
IFN-γ resistance of melanoma cells associated with low levels of HLA class I APM component expression. mRNA and protein expression patterns of HLA class I APM components in melanoma cells either left untreated or treated for 24 to 48 hours with IFN-γ were determined by qRT-PCR and Western blot analysis as described in Materials and Methods by using APM-specific primers and antibodies, respectively. A, LMP10; B, TAP2; C, HLA class I HC; D, tapasin. At least 3 independent experiments were carried out. The results are either expressed as mean of the values obtained in 3 independent experiments (qRT-PCR) or shown by a representative Western blot.
- Figure 3.
Association of impaired JAK2 expression in Colo 857 cells with impaired STAT1 phosphorylation. Melanoma cells were either left untreated or treated with IFN-γ for various time points before being harvested for protein extraction. Western blot analysis was carried out using unphosphorylated and phosphorylated JAK/STAT pathway component-specific antibodies. Staining of the Western blot with an anti-GAPDH- or β-actin–specific antibody served as controls.
- Figure 4.
Genomic deletion of JAK2 in Colo 857 cells. A, genomic PCR using JAK2-specific primers showed no amplification product in Colo 857 but in Colo 794. B, locus-specific gene amplification (end point PCR) was carried out as described in Materials and Methods. The RCC cell line MZ 2733RC known to have no observed abnormalities in IFN-γ signaling pathway served as a control (C) CGH. The deletion of JAK2 was determined by 3 independent technologies as described in detail in Materials and Methods.
- Figure 5.
Reconstitution of HLA class I APM component expression by Colo 857 cells following JAK2 gene transfer. A, JAK2 and pSTAT1 expression were determined in untreated and IFN-γ–treated Colo 857 cells transfected with the JAK2 expression vector as described in Materials and Methods. The results showed restoration of JAK2 expression and pSTAT1 upregulation by JAK2 gene transfer. B, constitutive and IFN-γ–inducible upregulation of APM component expression in mock-transfected and JAK2-transfected Colo 857 cells was carried out as described in Materials and Methods by using APM-specific mAbs. C, restoration of constitutive HLA class I surface expression in Colo 857 cells. Flow cytometry of untransfected, mock, and JAK2-overexpressing cells was carried out as described using an anti-HLA class I–specific mAb. The results are expressed as MFI with subtraction of the isotype control.
Tables
- Table 1.
Primers used for RT-PCR analysis
Application Gene name Sequence Tm IFN-γ signaling JAK1 fw: 5′-gca cca tca ccg ttg atg ac-3′ 60°C rev: 3′-tcc agt gag ctg gca tca ag-5′ JAK2 fw: 5′-tgt gga gat gtg ccg gta tg-3′ 60°C rev: 3′-att acg ccg acc agc act gt-5′ STAT1 fw: 5′-atc ctc gag agc tgt cta-3′ 55°C rev: 3′-gcc agg tac tgt ctg att-5′ Antigen processing LMP2 fw: 5′-tgt gca ctc tct ggt tca gc-3′ 60°C rev: 3′-tgc tgc atc cac ata acc at-5′ LMP10 fw: 5′-ggg ctt ctc ctt cga gaa ct-3′ 60°C rev: 3′-cag ccc cac agc agt aga tt-5′ TAP1 fw: 5′-gga atc tct ggc aaa gtc ca-3′ 60°C rev: 3′-tgg gtg aac tgc atc tgg ta-5′ TAP2 fw: 5′-cca aga cgt ctc ctt tgc at-3′ 60°C rev: 3′-ttc atc cag cag cac ctg tc-5′ Tapasin fw: 5′-tgg gta agg gac atc tgc tc-3′ 60°C rev: 3′-acc tgt cct tgc agg tat gg-5′ HLA-ABC fw: 5′-gcc tac gac ggc aag gat tac-3′ 60°C rev: 3′-ggt ggc ctc atg gtc aga ga-5′ Locus specificity RFX3 fw: 5′-aaa ctg gac cca gtc aat gc-3′ 57°C rev: 3′-tgt tgc atg ggt tgt tgt ct-5′ RCL1 fw: 5′-tct tct ttg ctt ggc tcc at-3′ 57°C rev: 3′-atg ggc ttc aag acc ttc ct-5′ GLDC fw: 5′-tcg atg cag ttc acc tca ag-3′ 57°C rev: 3′-cag att ccc acc tga gca tt-5′ BNC fw: 5′-gtc aag cat gcc tgt gaa ga-3′ 59°C rev: 3′-ggc cct cct tat ttc agt cc-5′ Control GAPDH fw: 5′-cct gca cca cca act gct ta-3′ 60°C rev: 3′-ctg aag gcc atg caa gtg ag-5′ PPIA fw: 5′-cca tct atg ggg aga aat ttg a-3′ 60°C rev: 3′-cag tca gca atg gtg atc ttc t-5′ β-Actin fw: 5′-tcc tgt ggc atc cac gaa act-3′ 60°C rev: 3′-gaa gca ttt gcg gtg gac gat-5′ Abbreviations: fw, forward; rev, reverse.
Supplementary Data
Supplementary Figures S1-S2.
Files in this Data Supplement:
Volume 17, Issue 9
