Fig 2: (A) Dose–response curves measuring infarct volume in vehicle-treated and 0.03, 0.1, 0.3, 3, and 10 mg/kg of decernotinib-treated mice. There was no significant difference in infarct size across groups (vehicle, n = 8; 0.03 mg/kg, n = 6; 0.1 mg/kg, n = 8; 0.3 mg/kg, n = 8; 3 mg/kg, n = 10; and 10 mg/kg, n = 8). Data were analyzed using a one-way ANOVA with a Dunnett’s multiple comparison post-test. (B) Representative 2,3,5-triphenyl-tetrazolium chloride (TTC) images of vehicle- and 10 mg/kg decernotinib-treated mice to show extent of cortical injury in our model of permanent middle cerebral artery occlusion (MCAO). (C) Representative immunoblots of brain tissue obtained from the ipsilateral (CXI) and contralateral (CXC) cerebral cortex depicting reduced phosphorylation of Janus kinase 3 (JAK3) in the 10 mg/kg decernotinib group compared to vehicle. There is no effect of decernotinib on stroke-induced increase in total JAK3 levels. (D,E) Graphical summary of immunoblot data (*P < 0.05 compared to vehicle ipsilateral; vehicle, n = 9; 10 mg/kg decernotinib, n = 5). Data were analyzed using a two-way ANOVA. (F) Latency to remove the sticky tape from the contralateral paw, measuring sensorimotor deficits, in vehicle and decernotinib groups. Stroke induced a significant increase in the latency to remove the sticker at 24 h in the vehicle-treated group. Comparing both treatments at 24 h, there was a trend toward a better performance in the adhesive removal test in mice given decernotinib, but this effect did not reach a statistical significance (two-way ANOVA with Bonferroni post-test; vehicle n = 9; decernotinib at a dose of 10 mg/kg, n = 10).

Fig 3: MiR-223 upregulates the expression of JAK2/STAT1 in NP cells. (a) The ratio of p-JAK1/p -JAK2/p-JAK3 to the internal reference. (b) WB band of p-JAK1/p-JAK2/p-JAK3. (c) Real-time manifestation of STAT1-STAT6 mRNA. ∗P < 0.05 vs. control; ∗∗P < 0.01 vs. control.

Fig 4: DHA treatment inhibited the HIF‐1α and JAK3/STAT3 signaling pathway in macrophages with LPS stimulation. (A–F) The expression of HIF‐1α, JAK3, p-JAK3, STAT3, and p-STAT3 at 24 h after incubation with or without LPS (100 ng/ml) and DHA (0.1, 0.2, and 0.4 μM). *p < 0.05 vs. the model group. #p < 0.05 vs. the control group.

Fig 5: Clinical sequencing informs clinical trial enrollment and experimental discoveryThe PHIAL output and treatment course for a patient with metastatic lung adenocarcinoma is shown, with the integration of clinical WES occurring during the patient’s first-line therapy allowing subsequent clinical trial enrollment (A). The patient’s time to relapse data for the three treatment regimens received demonstrate that the best and only clinical response occurred with the CDK4 inhibitor (B). Radiographic imaging demonstrates a small reduction in a representative metastatic focus for the patient on the CDK4 inhibitor trial after two cycles of therapy consistent with stable disease (cm: centimeter; measurement is 1.7 × 1.5 cm for baseline mass and 1.3 × 1.3 cm for two month interval scan of the same mass). Per RECIST criteria, overall tumor reduction was 7.9% (C). For another patient, PHIAL nominated a JAK3 missense mutation (D), and given its location in the kinase domain near alterations previously defined as activating, was considered to have inferential evidence (Level E) for being clinically actionable. The crystal structure of JAK3 demonstrates that the arginine at residue 870 directly coordinates the phosphate group of the primary activating tyrosine phosphorylation site (E). To better characterize this alteration, experimental follow-up of this alteration was performed in a Ba/F3 system. Overexpression of the patient’s JAK3 mutation did not suggest an activating phenotype or further consideration of JAK3 inhibitor clinical trial enrollment (F).