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. 2022 Mar 8;219(4):e20211273. doi: 10.1084/jem.20211273

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© 2022 Martin-Fernandez et al.

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Figure S3. — The I60N mutation leads to increased ISG mRNA and protein levels. (A–D) hTert-immortalized fibroblasts from control donor or P1 were treated with 1,000 IU/ml IFN-α2b for 8 or 24 h. Relative mRNA levels were assessed for IFIT1, MX1, RSAD2, and USP18, performed three times each with technical triplicates. A representative experiment is shown. Data analysis was performed with unpaired t tests. ns, P > 0.05; *, P < 0.01; **, P < 0.001; ***, P < 0.0001. (E–G) hTert-immortalized fibroblasts from control donor (C1 WT/WT), P1 (I60N/I60N), mother (WT/I60N), and healthy brother (C2 WT/WT) were treated with 1,000 IU/ml IFN-α2b for 12 h, washed, and left to rest for 36 h. Relative mRNA levels were assessed for IFIT1, MX1, and RSAD2, performed three times each with technical triplicates; representative experiment shown. Statistical analysis performed by one-way ANOVA. ***, P < 0.0001. (H and I) hTert-immortalized fibroblasts from a control donor (Control), P1 (I60N), or USP18-deficient patient (P2) were treated with IFN-α2b (100 pM) for the indicated times. Cell lysates were analyzed by Western blot for the indicated antibodies; representative experiment shown. (J) hTERT-immortalized fibroblasts from a control donor (Control), P1, or P1 lentivirally transduced with WT-USP18 were primed with IFN-α2b (500 pM) for 8 h, washed, left to rest for 36 h, and restimulated with the indicated doses of IFN-α2b for 20 min. Cell lysates were analyzed by Western blot with the indicated antibodies; a representative experiment is shown. (K) hTert-immortalized fibroblasts from a control donor (Control), P1, or P1 lentivirally transduced with WT-USP18 were treated with IFN-α2b (1,000 U/ml) for the indicated times. Cell lysates were analyzed by Western blot for the indicated antibodies; representative experiment shown. (L–N) hTert-immortalized fibroblasts from a control donor (Control) or P1 were mock-transduced (−) or transduced with Luc-RFP (Luc) or WT USP18 (WT), sorted, treated with the indicated doses of IFN-α2b for 12 h, washed with PBS, and left to rest for 36 h, after which relative mRNA levels were assessed for the genes indicated, performed three times each with technical triplicates; representative experiment shown. Bars represent the mean ± SD. (O) hTert-immortalized fibroblasts from Control untransduced (−) or USP18-deficient patient untransduced (−) or transduced with Luc or with USP18-WT or -I60N and sorted. Cells were treated with the indicated doses of IFN-α2b for 12 h, washed with PBS, and left to rest for 36 h, after which relative mRNA levels were assessed for MX1, performed three times each with technical triplicates; representative experiment shown. Bars represent the mean ± SD. R.U., relative units. All results are representative of at least two independent experiments. The molecular weight markers (kD) are shown on the left.