Fig. 2: Monobody inhibition of STAT3 transcriptional activity.

a Scheme depicting the initial screening strategy to identify monobodies with STAT3 inhibitory activity. b Luciferase reporter activity inductions normalized to that of unstimulated cells, which was arbitrarily set to 1, are reported for the expression of the indicated monobodies and control treatments. Results from two individual experiments performed in triplicate and/or duplicate are shown (data presented as mean ± SD, n = 5 or 6). Two-tailed unpaired t-test analysis was performed against the HA4-Y87A monobody control: MS3-6 **P = 0.0027, Ruxolitinib *P = 0.0158, siRNA JAK1 *P = 0.0147, STAT3-Y705F *P = 0.028. c BW5147 or Ba/F3 cells (10⁷) were electroporated with the monobody plasmid, the pGL3-Pap1 luciferase reporter plasmid as a specific promoter for STAT3 activation and the pRL-TK plasmid. Cells were stimulated with control medium or murine IL-9 (100 ng ml⁻¹, left upper panel, n = 11), human IL-22 (500 ng ml⁻¹, right upper panel, n = 12), human IL-24 (HEK293 supernatant 2%, lower left panel, n = 9) or human IFNλ3 (HEK293 supernatant 2%, lower right panel, n = 6) for 4 h. Data are presented as mean ± SEM from two to four independent experiments performed in triplicates. d Luciferase assay on A549 with inducible monobody (HA4-Y87A or MS3-6) expression. A549 cells (5 × 10³) were plated in 96-well plate and treated for 24 h with control medium or doxycycline (1 µg ml⁻¹). Cells were transiently transfected with the pGL3-Pap1 luciferase reporter plasmid and the pRL-TK plasmid as internal control of transfection. In all, 4 h after transfection, cells were stimulated with control medium or human IL-6 (100 ng ml⁻¹, upper panel) or human IL-22 (500 ng ml⁻¹, lower panel) for 20 h. Data are presented as mean ± SEM of three independent experiments performed in triplicates. Significance in c, d is shown according to two-tailed Mann–Whitney test: *P = 0.026, ****P ≤ 0.0001. e RT-qPCR for expression of STAT3 downstream genes in A549 cells expressing monobodies upon IL-22 stimulation (100 ng ml^-1, 1 hr at 37 °C). Gene expression was normalized to actin and is presented as fold change against that of untreated A549 cells (no monobody expression), which was set to 1. Data from three independent experiments performed in triplicates (n = 9) are presented as mean ± SD. Significance was calculated against mRNA levels in the HA4-Y87A monobody control conditions. Significance is shown according to two-tailed unpaired t-test analysis: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are provided as a source data file.