Fig 2: Protein expression levels of leptin signaling pathway components and PTP1B. The protein expression levels of leptin signaling pathway components and PTP1B in the hypothalamus. An endogenous control (β-actin) was used to normalize the protein expression. (A) The protein levels of PTP1B in the RYGB group were significantly lower than those in the SO group (P<0.05), and similar to those in the Lean group. (B) The expression levels of p-STAT3 in the RYGB group were significantly higher than those in the SO group (P<0.05) and similar to those in the Lean group, while the expression of STAT3 was similar in all groups. *P<0.05, RYGB vs. SO; #P<0.05, ##P<0.01, SO vs. Lean. p-STAT3, phosphorylated signal transducer and activator of transcription; PTP1B, protein tyrosine phosphatase 1B; SO, sham operation; RYGB, Roux-en-Y gastric bypass.

Fig 3: Activation of the RXFP1‐JAK3‐STAT3‐Cdc42 axis increases GBM motility. Representative real‐time migration curves of GBM10 motility measured by the RTCA‐DP system using CIM plates. Experiments were performed for up to 25 h, and measurements were recorded every 15 min. Cumulative measurement graphs (15–25 h) showed an average of 70% induction of GBM10 motility upon CTRP8 treatment (B, D, J, L). The enhanced migratory response of patient GBM cells to CTRP8 was abolished in the presence of siRXFP1 (A, B) and siCdc42 (C, D). The siCdc42 treatment selectively and significantly reduced GBM cell viability as measured by real‐time RTCA‐DP system using E‐plates. This effect was not observed with control siRNA. Treatment with siCdc42 was nontoxic and did not affect cell viability (E, F). Silencing of RXFP1 in GBM10 cells did not change the level of cell viability as measured by RTCA‐DP system. Treatment with control siRNA had no effect on GBM cell migration (A–D) or viability (G, H). Treatment with JAK1/3 inhibitor tofacitinib (10 µm; I, J) and STAT3 inhibitor S3I‐201 (30 µm; K, L) abolished the CTRP8‐mediated increase in GBM motility. Each experiment was performed as three independent duplicates for each treatment. ANOVA was used to determine significance by calculating four measurements for each experiment; results are displayed using graphpad prism. Data are represented as a mean values and SD with a P‐value of <0.001 (***) and < 0.00001 (****); NS, not significant.

Fig 4: STAT3 activity contributes to IL‐10 production by CD19+ Tim‐1+ cells. (A) STAT3 mRNAs of CD19+ Tim‐1+ cells from both mice were measured by reverse transcription and real‐time PCR. (B) Total STAT3, STAT3‐pY705, and STAT3‐pS727 levels were determined by Western blot. (C) Statistical analysis of STAT3 expression in CD19+ Tim‐1+ cells from both mice. Variations of IL‐10 (D) and Tim‐1 (E) expression in CD19+ Tim‐1+ cells treated with the STAT3 inhibitor (Stattic). Each experiment was repeated at least thrice. *P < 0.05; ***P < 0.001

Fig 5: CTRP8 utilizes a new RXFP‐JAK3‐STAT3 axis for increased Cdc42 protein and activity in GBM cells. Western blot detection of Cdc42 in patient GBM10 (A), GBM34 (C), and U87MG (E) cells upon 24‐h stimulation with CTRP8 in the presence and absence of siRXFP1, siCdc42, JAK3 inhibitor tofacitinib, and STAT3 inhibitor S3I‐20. In all three GBM models, CTRP8 treatment increased the cellular Cdc42 protein content by approx. 40–50% compared with nontreated control, as quantified by densitometry of eight independent experiments for each model (n = 8), including GBM10 (B), GBM34 (D), and U87MG (F). Densitometry graphs represent mean with SD. Two‐tailed t‐test was used to determine the P‐value, and P < 0.05 was considered significant, and P‐values are indicated. qPCR analysis was used to show successful siRXCFP1‐mediated reduction of RXFP1 transcripts (G). Western blot detection of Cdc42 showed successful reduction in Cdc42 protein upon siCdc42 treatment (G). Detection of GTP‐bounded (active) Cdc42 in U87MG and GBM10 cells (H). GBM10 and U87MG cells treated with CTRP8 for 0–60 min were incubated with GST‐PAK1‐PDP, and PAK1‐interacting partners were pulled down with glutathione resins at a total protein input of 500 μg. Employing a specific Cdc42 antibody, we detected significant levels of PAK1‐interacting partner GTP‐Cdc42 at 10 and 20 min in GBM10 and U87MG by western blot (H). Western blot detection of pJAK3Tyr980/981, total JAK3, pSTAT3Tyr705, and total STAT3 in GBM10 upon CTRP8 treatment for 0, 5, 10, 20, 30, and 60 min (I–L). Phosphorylation of both JAK3 and STAT3 was diminished in GBM cells treated with CTRP8 and siRXFP1 KD (I), whereas phosphorylation of JAK3 and STAT3 was observed upon siCdc42 treatment (J). GBM cells co‐treated with CTRP8 and tofacitinib showed diminished pJAK3Tyr980/981 and pSTAT3Tyr705 phosphorylation (K), while co‐treatment with CTRP8 and S3I‐201 abolished STAT3 phosphorylation but failed to inhibit JAK3 phosphorylation (L). Our results identified a new CTRP8‐RXFP1‐JAK3‐STAT3‐Cdc42 axis. Beta‐actin was used as a loading control in all western blots (A, C, E, G, H–L).