Fig 2: MPXV protein D8L inhibits IFN-induced signaling. a HEK-293T cells were transfected with pcDNA3.1(+)-D8L-mCherry-FLAG plasmid (0 µg, 0.65 µg, 1.3 µg, and 2.6 µg), PEF2-IFNλ1-FLAG (0.2 µg), reporter plasmid ISRE-Luc (1 µg), plus TK-Renilla (0.2 µg). The EV plasmid was used to standardize the amount of plasmid transfected in each well. After 24 h, the red fluorescence signal of the pcDNA3.1(+)-D8L-mCherry-FLAG plasmid was observed by using an inverted fluorescence microscope, after which the luciferase and Renilla activities were determined. The lysates were analyzed by WB to verify the accurate expression of the plasmids. b HEK-293T cells were transfected with pcDNA3.1(+)-D8L-mCherry-FLAG plasmid (0 µg, 0.65 µg, 1.3 µg, and 2.6 µg), reporter plasmid ISRE-Luc (1 µg), plus TK-Renilla (0.2 µg). The EV plasmid was used to standardize the amount of plasmid transfected in each well. After 24 h, HEK-293T was transfected with pCMV-IFNB1-FLAG plasmid (2 µg) for 6 h. Then, the luciferase and renilla activities were measured. The lysates were analyzed by WB to verify the accurate expression of the plasmids. c In the experiment to detect the IFN RNA levels, the pcDNA3.1(+)-D8L-mCherry-FLAG plasmid 2 µg or the EV plasmid 2 µg) was transfected into HEK-293 T. After 24 h, the Poly(dA: dT) (2 µg/mL) was transfected into HEK-293T. After 12 h, the mRNA transcript levels of IFN-β and IFN-λ of total RNA extracted from the cells were assessed by RT-qPCR. Data were expressed as fold change with GAPDH as the internal control. In the experiment to detect the ISGs RNA levels, the pcDNA3.1(+)-D8L-mCherry-FLAG plasmid (1.5 µg) and PEF2-IFNλ1-FLAG plasmid (0.5 µg) were transfected into HEK-293T. The EV plasmid was used to standardize the amount of plasmid transfected in each well. After 24 h, the mRNA transcript levels of Cig5, IFITM1, MX2, OAS1, ISG56, and ISG15 of total RNA extracted from the cells were assessed by RT-qPCR. Data were expressed as fold change with GAPDH as the internal control. NS: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t-test. d The pcDNA3.1(+)-D8L-mCherry-FLAG plasmid (1.5 µg) and PEF2-IFNλ1-FLAG plasmid (0.5 µg) were transfected into HEK-293T. The EV plasmid was used to standardize the amount of plasmid transfected in each well. The cells were lysed after 24 h, and the IFIT1, IFITM1, and ISG15 expressions in the lysates were analyzed by WB

Fig 3: IFNβ treatment phenocopies several aspects of chronic cisplatin treatment and drives therapy resistance. (A) The IC50 of SE and CR cells was significantly higher following IFNβ treatment. (B) Morphology change was determined by measuring the aspect ratio (width/legth) of 60 cells from each sample (20 cells per photo, 2 photos per well, 3 wells). Scale bar = 50 μm. (C) BrdU staining revealed cell cycle arrest in G0‐G1 following IFNβ treatment, as measured by flow cytometry. Data represent one representative replicate. n = 2. (D) Proliferation was detected by counting individual wells of parental and IFNβ treated cells over time. (E) RT‐qPCR was used to analyze STAT1, IFITM1, and PLSCR1 following 6 passages and 12 weeks following consistent low‐level IFNβ treatment. (F) Intracellular flow cytometry was used to measure STAT1 and pSTAT1 protein levels following chronic IFNβ treatment. Individual replicates normalized to average control samples. Results are displayed as n = 3, unless otherwise noted, and presented as the mean ± SD. Statistical significance was determined using the unpaired t‐test. P‐values: P ≤ 0.05 (*) and P ≤ 0.01 (**).

Fig 4: The IFN‐I pathway is activated in CR cells. (A) RT‐qPCR analysis of IFN‐1 RNA levels. (B) Secreted IFN‐1 protein levels detected in culture supernatant via HEK‐Blue SEAP reporter assay. (C) RT‐qPCR analysis of IRDS genes. Dots indicate genes known to be transcribed by u‐ISGF3. (D) Intracellular flow cytometry of IFITM1, total STAT1, and pSTAT1(y701). One representative replicate shown. Results are displayed as n = 3, unless otherwise noted, and presented as the mean ± SD. Statistical significance was determined using the unpaired t‐test. P‐values: P ≤ 0.05 (*).