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. 2016 Oct 1;27(10):847–859. doi: 10.1089/hum.2016.065

Figure 2.

Figure 2.

Perforin expression in the human KHYG1 NK cell line from LV vectors carrying the MND, PGK, and PRF promoters. (a) Bidirectional LV vector design with a mini-CMV promoter driving the human tNGFR cDNA in one direction, and (i) MNDU3 promoter (MND4T), (ii) tissue-specific perforin gene promoter (PRF0T), or (iii) phosphoglycerate kinase (PGK4T) promoter driving expression of perforin in the opposite direction. miR126 target sequences (red vertical lines) were placed downstream of the woodchuck posttranscriptional regulatory element (WPRE) in the MND and PGK vectors. (b) Relative Prf1 mRNA expression in wild-type KHYG1 cells (WT), KHYG1 permanently transduced with prf1 shRNA to the 3′ UTR of the prf1 gene (KHYG1 KD), and KD cells transduced (and selected for tNGFR+) with the three LV vectors. Expression is normalized to KHYG1 KD mRNA expression (n = 3 experiments). (c) Representative fluorescence-activated cell-sorting (FACS) plots showing perforin versus tNGFR expression in wild-type and KD KHYG1 cells, and KHYG1 KD cells transduced with MND4T, PGK4T, or PRF0T LV vector; bar diagram shows the cumulative results of Prf1 mean fluorescence intensity (MFI) in these cells (n = 3 experiments). (d) Representative Western blot analysis showing relative perforin protein in KHYG1 WT, KD, and KD cells transduced with the three LV vectors. Numbers represent fold increase in expression over KD cells. (e) Cumulative data from Western blots showing fold increase in Prf1 protein expression compared with KHYG1 KD cells. Error bars represent the SEM. *p < 0.05.