Supplementary MaterialsS1 Fig: Hypoxia downregulates MHC class I expression in 3-dimensional (3D) however, not in 2-dimensional (2D) culture systems

Supplementary MaterialsS1 Fig: Hypoxia downregulates MHC class I expression in 3-dimensional (3D) however, not in 2-dimensional (2D) culture systems. spheroids cultured under normoxic circumstances. MCA-205 fibrosarcoma cells had been harvested as spheroids (S2A) or toned monolayer (S2B) in normoxic circumstances (21% air) and degrees of hypoxia in each lifestyle system evaluated using hypoxyprobe. About 16% of the populace was hypoxic in the 3D spheroids whereas there is no detectable degrees of hypoxia in the 2D cultured cells. In 3D spheroids of Un4, about 20% of the populace was hypoxic (S2D). Representative contour plots of three indie experiments proven. (S2C, E) Mean fluorescent strength (MFI) of MHC course I appearance on 3D spheroids (MCA205; S2C, Un4; S2E) from much less hypoxic (HP low) and even more hypoxic (HP high) locations showed inverse relationship between hypoxia and MHC course I expression. Each stage on graph represents an unbiased test with the common symbolized as a dash.(TIF) pone.0187314.s002.tif (581K) GUID:?9BDC286E-7D4A-4624-8694-4AB63F2F1460 S3 Fig: Hyperoxia upregulates MHC class I expression equally in 2D and 3D cultures. 4T.1 breast carcinoma Ononin (S3 A,B), P815 mastocytoma (S3 C,D), RMA T lymphoma (S3 E,F) and EL4 Ononin thymoma (S3 G,H) were cultured as 2D monolayers (indicated as 2D) or as 3D spheroids (indicated as 3D) and cultured under 21% O2 or 60% O2 for 48h. Levels of MHC class I expression was decided using flow cytometry. Representative histograms of 4 impartial experiments are shown. Grey packed: unstained control; blue: normoxia; green: hyperoxia. MFI: mean fluorescence intensity.(TIF) pone.0187314.s003.tif (469K) GUID:?6162D53C-08C3-499E-844B-BA24A92DD304 S4 Fig: Hypoxia downregulates MHC class I expression via HIF transcription factors; extended data from Fig 6. (SA, B): siRNA mediated knockdown of HIF-1 reversed hypoxic downregulation of MHC class I expression as compared with the scrambled, non-targeting (NT) siRNA control. MCA205 tumor cells were reverse transfected with scrambled siRNA (NT) or with HIF-1 specific siRNA and cultured as 3D spheroids under 1% or 21% oxygen for 48h. Levels of MHC class I surface expression was decided using flow cytometry; quantitative analysis of representative histograms shown in Fig 6 shown here (A). Levels of MHC class I transcripts were assessed using RT-qPCR (B). Average data of 3 impartial experiments are shown.(S4C, D): Flow cytometry assessment of surface expression of HLA-ABC (S4C) and RT-qPCR analysis of HLA-ABC transcript levels (S4D) on paired isogenic renal cell carcinoma cell lines RCC4, UMRC2 and CAKI2. Each pair had the parental cell line that lacked endogenous wild-type VHL (VHL null, transfected with vacant vector) and one with vector stably expressing functional VHL (VHL restored). Restoring VHL function and thereby reducing HIF expression, significantly increased HLA-ABC surface expression and transcript levels in the cells. Average data of 4 impartial experiments are shown. (TIF) pone.0187314.s004.tif (236K) GUID:?21ECDF54-42BC-4153-A21C-9DC8F81405C1 S5 Fig: Hypoxia downregulates MHC Class I expression via Hif-1(S5A-D). siRNA mediated knockdown of HIF-1 reversed hypoxic downregulation of MHC class I expression as compared with the scrambled, non-targeting (NT) siRNA control. EL4 tumor cells were reverse transfected with scrambled siRNA (NT; Red histogram) or with HIF-1 specific siRNA (blue histogram) and cultured as 3D spheroids under 1% (S5A) or 21% (S5B) oxygen for 48h. Levels of MHC class I surface expression was decided using flow cytometry (S5A,B). RT-qPCR was used to analyze MHC class I transcript levels. Ribosomal protein L32 was used as internal control (S5C). Efficacy of gene knockdown was assessed using western blot (S5D). -actin was used as the loading control. Representative data of 2 impartial experiments are shown.(PNG) pone.0187314.s005.png (222K) GUID:?1AFC85FC-2411-417F-92B0-4452816EA257 S6 Fig: HIF-1 and HIF-2 have redundant roles in downregulating MHC class I expression. (S6A-C): siRNA mediated knockdown of HIF-1, HIF-2 or both reversed hypoxic downregulation of MHC class I expression MADH3 as compared with the scrambled, non-targeting (NT) siRNA control. MCA205 tumor cells were reverse transfected with scrambled siRNA (NT) or with HIF-1, HIF-2 or both HIF-1 and HIF-2 specific siRNA and cultured as 3D spheroids under 1% or 21% oxygen for 48h. Levels of MHC course I Ononin surface appearance was motivated using movement cytometry (S6A). Transcripts amounts.