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. 2023 Sep 21;8(20):e169344. doi: 10.1172/jci.insight.169344

Figure 2. TLR7 ligands in XIST activate human pDCs and HEK-hTLR7 cells.

Figure 2

(A) Sequences of the 4 RNA oligonucleotides used to stimulate pDCs and HEK-hTLR7 cells. (B) ELISA measuring IFN-α production by pDCs from 4 different healthy donors after transfection with 20-mer oligonucleotides. (C) Colorimetric assay showing the production of SEAP by HEK-hTLR7 cells in response to treatment with 20-mer oligonucleotides in 3 technical replicates. All treatments were compared with XIST1.1 by multiple comparisons at each dose. † Indicates XIST1.1 versus RNA9.2s, # indicates XIST1.1 versus RNA9.2a, and * indicates XIST1.1 versus polyA. One symbol indicates P < 0.05, 2 symbols indicate P < 0.01, 3 symbols indicate P < 0.001, and 4 symbols indicate P < 0.0001. (D) Fluorescence polarization values of XIST1.1, RNA9.2s, RNA9.2a, and polyA RNA when incubated with increasing doses of human recombinant TLR7. (E) ELISA measuring IFN-α production by pDCs after transfection with A-repeat or control RNA from 4 different healthy donors. IMQ was used as a positive control. (F) Colorimetric assay showing the production of SEAP by HEK-hTLR7 cells after transfection with A-repeat or control RNA in 6 technical replicates. (G) ELISA measuring IFN-α production by pDCs after transfection with A-repeat with or without TLR7 inhibitors HCQ or ODN from 3 different healthy donors. (H) ELISA measuring IFN-α production by pDCs after transfection with varying concentrations of A-repeat RNA or XIST1.1 in 3 technical replicates. A-repeat was compared to XIST1.1 by t test at each dose. (B–H) Error bars indicate 1 standard deviation. * Indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. (B and E–G) All treatments were compared with XIST1.1 or A-repeat by 1-way ANOVA with multiple comparisons correction.