Fig 2: TA prevents IL-6/EGF-induced tyrosine phosphorylation of STAT3. (A, C) Cells were stimulated with different concentrations of IL-6 (A) or EGF (C) for 30 min, p-STAT3/STAT3 were detected. (B, D) Cells were pretreated with TA for 12 h and stimulated with IL-6 (B) or EGF (D) for 30 min, cell lysates were subjected to Western blotting to determine the p-STAT3/STAT3 level. (E) The DNA-binding activity of STAT3 was detected by EMSA. (F) U2OS cells were treated with indicated concentrations of TA, p-JAK1/2/3, and total JAK1/2/3 were examined by Western blotting. (G) U2OS cells were treated with the indicated concentration of pervanadate for 30 min, followed by 2 µM TA for 24 h, and the cell lysates were subjected to Western blotting. (H) The cells were treated with or without TA for 24 h and cell lysates were subjected to IP assay to determine the PTP activity of SHP-1, SHP-2, PTEN, and PTP1B, *P < 0.05, ***P < 0.001 versus control. (I) U2OS cells were transfected with negative control siRNA or SHP-1, SHP-2, PTEN, and PTP1B specific siRNA (100 nM). After 24 h, cells were treated with 2 µM TA for 24 h. Whole-cell extracts were prepared and immunoblotted with p-STAT3/STAT3 antibodies. (J) U2OS cells were pretreated with 25 µM NSC-87877 for 30 min, followed by 2 µM TA treatment for 24 h. Cell lysates were prepared and subjected to western blotting analysis.

Fig 3: Studies of potential interactions between Zn27 and the tyrosine phosphatases that inactivate FAK. (A) Zn27 (10 nM) stimulated FAK-Tyr-397 phosphorylation as measured by Western blot in Caco-2 cells without or with PTP LYP inhibitor (50 µM) (n = 25, *p < .05). (B) Densitometric analysis of the blots described in (A) Because PTP LYP inhibition dramatically accentuated FAK phosphorylation, a Y-axis break has been placed to be able to visually represent the effects of Zn27 on FAK phosphorylation in both the absence and presence of PTP LYP inhibitor on the same graph. (C) Lack of effect of 10 nM Zn27 on FAK-PTP1B association in suspended Caco-2 cells. Cell lysates were immunoprecipitated with anti-FAK and then blotted for either co-precipitating PTP1B (top panel) or FAK itself (bottom panel). Input is shown in the first two lanes and the immunoprecipitant in the third and fourth lanes. (D) Densitometric analysis of eight similar studies to the one shown in C demonstrates that treating with Zn27 (10 nM) does not affect the amount of PTP1B co-precipitating with FAK (n = 8) (E) Lack of effect of Zn27 (100 nM) on SHP2 activity (n = 2). All data are represented as mean ± SE, *p < .05

Fig 4: Xrn1-cKO mice exhibit upregulated appetite and energy homeostasis-related genes in the hypothalamus(A) Quantitative PCR (qPCR) analysis of the indicated mRNA levels in the hypothalamus of 10- to 14-week-old control (Xrn1flox/flox: white, n = 5; Camk2a-Cre: gray, n = 10) and Xrn1-cKO mice (red, n = 5). The value of the average of Xrn1flox/flox mice was set as 1. Data represent means ± SEM. One-way ANOVA, Tukey post hoc test, *p < 0.05; **p < 0.01; and ***p < 0.001.(B) qPCR analysis of the indicated pre-mRNAs in the hypothalamus of 10- to 14-week-old control (Xrn1flox/flox: white, n = 5; Camk2a-Cre: gray, n = 10) and Xrn1-cKO mice (red, n = 5). The average value of Xrn1flox/flox mice was set as 1. Data represent means ± SEM. One-way ANOVA, Tukey post hoc test, *p < 0.05; **p < 0.01; and ***p < 0.001.(C) Protein expression of XRN1, LERP, INSR, STAT3, FOXO1, SOCS3, PTP1B, TCPTP, and a-Tubulin in 12-week-old control (Xrn1flox/flox: white, n = 3; Camk2a-Cre: gray, n = 3) and Xrn1-cKO mice (red, n = 3).(D) Quantitative analysis of protein bands in panel 5C. Band intensities were measured using NIH ImageJ. Values of the average of Xrn1flox/flox mice were set as 1. Data represent means ± SEM. One-way ANOVA, Tukey post hoc test, *p < 0.05; **p < 0.01; and ***p < 0.001.(E) Immunofluorescence analysis of AgRP (red) and nuclei by counterstaining with DAPI (blue) in the ARC of Xrn1-cKO and control (Xrn1flox/flox) mice at 12 weeks old.(F) Quantification of the AgRP signal in the ARC of Xrn1-cKO (red) and control (white) mice.

Fig 5: NKX2-1-AS1 knockdown alters gene expression patterns in H441 cells. (A) List of the top 20 genes down- and (B) up-regulated by NKX2-1-AS1 knockdown determined by microarray analysis, 48 h after treatment (n = 6). (C) qPCR validation of down-regulated and up-regulated genes in NKX2-1-AS1 knockdown cells at 48 h after treatment (n = 3, *p < 0.05, **p < 0.01). (D) Real time PCR validation of adherens junction related genes up-regulated by NKX2-1-AS1 knockdown (n = 3; *p < 0.05; **p < 0.01). (E) Representative western blots of PD-L1 and PTPN1 protein in non-silencing control [c] and NKX2-1-AS1 siRNAs [si] treated H441 cells normalized to ß-actin. (F) Densitometry of the western blot signals normalized to ß-actin indicates that PD-L1 (n = 9; **p = 0.002) and PTPN1 (n = 3; *p = 0.05) protein levels are increased by NKX2-1-AS1 knockdown.