Fig 2: Tcf21 inhibits Tcf21 LC adipogenic efficiency through promoting Dlk1 expression(A) Tcf21 LCs from eVAT of KO mice of the indicated groups were subjected to bulk RNA-seq and PCA. Samples of the same color indicate littermates.(B) A bar graph shows Tcf21 expression in the indicated groups. n = 5 for 3 weeks; n = 2 for 1 week HFD and 12 weeks HFD; paired t test. Data are represented as mean with values of littermates connected.(C) A heatmap shows the expression of DEGs between Tcf21 LCs of WT and KO 3-week mice in cells of the indicated groups.(D and E) Graphs show the NES (normalized enrichment score) of representative BPs enriched in DEGs that were upregulated (D) or downregulated (E) in 3-week WT versus KO mice.(F) Tcf21 LCs isolated from eVAT of the indicated groups were subjected to ATAC-seq and PCA. Samples of the same color indicate littermates.(G–I) The absolute LFC in promoter (G) and distal region (H) accessibility of DEGs between Tcf21 LCs of 3-week WT and KO mice are plotted. A pie graph (I) shows the numbers of DEGs with corresponding DAPs, DADs, DAPs and DADs, or none. See also Table S6.(J) The enrichment of the Tcf21 motif in ADRs in Tcf21 LCs of the indicated sample groups.(K) A GRN was constructed using direct target genes of Tcf21 and 2 Tcf21-targeted TFs. See also Table S7.(L) eVAT of P12 Tcf21 LT mice was subjected to IHC to identify Dlk1+ Tcf21 LCs (yellow arrows). Scale bar, 20 µm.(M) The expression of Dlk1 in eVAT Tcf21 LCs of the indicated groups was measured by RNA-seq. n = 5 for 3 weeks; n = 2 for 1 week HFD and 12 weeks HFD; paired t test.(N) The Integrative Genomics Viewer view shows the expression and chromatin accessibility of Dlk1 in Tcf21 LCs of the indicated sample groups.(O and P) SVCs isolated from 3-week C57BL/6 mice were transduced with IRES-GFP (Ctrl) or Dlk1-IRES-GFP (OE) retroviruses, verified for Dlk1 OE by real-time PCR and ICC (O), and induced for adipogenesis followed by LipidTOX staining (P). n = 3; unpaired t test. Scale bar, 20 µm.(Q) Tcf21 LCs sorted from 3-week WT and KO mice were transduced with Dlk1-IRES-GFP retroviruses and induced for adipogenesis, followed by LipidTOX staining. n = 3; unpaired t test. Scale bar, 20 µm.(R) Dpp4+ and Dpp4-;Icam1+ Tcf21 LCs isolated from 3-week WT and KO mice were induced for adipogenesis and stained with LipidTOX. n = 3; unpaired t test. Scale bar, 50 µm.*p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean with values of littermates connected (B and M) or mean ± SEM with individual values plotted (O–R). See also Figure S5 and Tables S6 and S7.

Fig 3: Forced expression of ß-catenin, YAP, and c-Met induced HB-like tumorigenesis in vivo. a HB tissues (n = 11) were stained with anti-YAP antibody and hematoxylin. Percentages of YAP-positive cases in the tumor lesions and nontumor regions are shown. b Tissue sections of the liver isolated from mice injected with ?Nß-catenin and YAPS127A (BY), ?Nß-catenin and c-Met (BM), or ?Nß-catenin, YAPS127A, and c-Met (BYM) at 6 weeks after hydrodynamic tail vein injection of plasmids, were stained with anti-GREB1, anti-DLK1, and hematoxylin. Solid squares showed the representative gross images of livers from mice injected with BY, BM, or BYM. c Total RNAs were prepared from liver tumor nodules isolated from BY (n = 3), BM (n = 7), or BYM (n = 11) mice. Real-time PCR experiments for GREB1 and TACSTD1 mRNA expression are performed. Results are expressed as fold changes compared with BY and are shown as dots plot with median. Scale bars in a and b (left six panels), 200 µm; in b (right three panels), 1 cm

Fig 4: Pref-1 regulates non-inflammatory MIF release from WAT which induces IR(A) TNF (TNF-a), IL1B (IL-1ß), and IL6 (IL-6) mRNA expression in biopsied WAT from male subjects including lean (age: 23.3 ± 2.2; BMI = 25 kg/m2) (N = 10) and obese individuals (age: 24.3 ± 3.3; BMI > 30 kg/m2) (N = 10).(B) HOMA insulin resistance scores (HOMA-IR scores) of recruited lean and obese individuals evaluated by plasma glucose and insulin levels.(C–F) Pref. 1 (Pref-1) (C), PPARG (PPAR?) (D), and MIF (E) mRNA expression in biopsied WAT and plasma MIF levels (F) from recruited lean and obese individuals.(G and H) The correlation between HOMA-IR scores and plasma MIF levels, and the correlation between Pref. 1 mRNA expression and plasma MIF levels.(I) Pro-inflammatory macrophage (M1 MF) markers: Tnfa (TNF-a) and Il1b (IL-1ß), and general inflammatory (MF, macrophages) marker: Il6 (IL-6) in visceral epididymal adipose tissue collected from WT mice (20-week-old) fed with normal chow (NC) or a high palmitic acid diet (PD) for 8 weeks.(J) The ratio of M1/M2 was evaluated by flow cytometry.(K) Insulin sensitivity was quantified by i.p. glucose tolerant test (GTT) and insulin tolerant test (ITT). (I and M) Adipose pref-1, FABP4 and PPAR? mRNA expression (L) and Pref-1 immunofluorescence staining (M) from WT mice (20-week-old) fed with NC or PD for 8 weeks (Arrows, Pref-1 (red); Scale bars, 20 µm).(N) Pref-1+ cells sorted by flow cytometry from SVFs (left) and Pref-1 release from visceral adipose tissues (right) isolated from NC and PD groups.(O–Q) MIF gene and protein expression in WATs isolated from NC and PD groups (Scale bars, 20 µm).(R) Plasma MIF levels in NC and PD groups.(S) MIF release from WATs isolated from NC and PD groups in the presence or absence of recombinant Pref-1 proteins (2.5 µg/ml).(T) Insulin sensitivity quantified by GTT and ITT in PD groups following MIF neutralization with anti-MIF antibody (20 mg/kg, i.p. twice a week).(U) Plasma MIF levels in 25-week-old WT and Mif lung Tg mice.(V) Pro-inflammatory macrophage (M1 MF) markers: Tnfa (TNF-a) and Il1b (IL-1ß), and general inflammatory (MF, macrophages) marker: Il6 (IL-6) in visceral epididymal adipose tissue collected from 25-week-old WT and Mif lung Tg mice.(W) Insulin sensitivity quantified by GTT and ITT in 25-week-old WT and Mif lung Tg mice.All data are presented as mean ± SD. #p = 0.05 reduction vs. lean subjects in (C), vs. NC group in (N), and (P); *p = 0.05 increase vs. lean subjects in (B) and (F), vs. NC group in (K) and (R), vs. WT group in (U) and (W), vs. other groups in (S) and (T). The n.s. represents no significance.

Fig 5: PAR2 deficiency in WAT or addition of Pref-1 decreases non-inflammatory adipose MIF release and improves insulin sensitivity(A) WT mice at 18 weeks were anesthetized and transplanted with WT or Par2-/- visceral epididymal adipose tissue. Following 2-week recovery, these mice at 20 weeks will be fed with normal chow (NC) or high palmitic acid diet (PD) for 8 weeks.(B–H) Plasma Pref-1 (B) and MIF (C) levels in the mice transplanted with WT or Par2-/- adipose tissue following NC or PD diet feeding. Insulin resistance was assessed by GTT and ITT (D). Body weight gain, adipose weight (AT)/body weight (BW) ratio, adipocyte size, and M1/M2 ratio in adipose tissue were measured in these mice as well (E to H) (Scale bars, 20 µm in G).(I) WT mice at 20 weeks were fed with NC or PD for 8 weeks. During the last 4 weeks, the mice were infused with vehicle or recombinant Pref-1 protein (rPref-1, 24 µg/day/kg) by osmotic pump.(J–R) Plasma Pref-1 levels, PPAR? gene expression, adipose MIF and p115 contents, and plasma MIF levels were subsequently evaluated from (J) to (M). Insulin resistance was assessed by GTT and ITT in (N). Body weight gain, adipose tissue weight, adipocyte size, and expression of inflammatory factors were quantified from (O) to (R) (Scale bars, 20 µm in Q).(S) Schematic diagram for the mechanism of PAR2/AMPK/Pref-1/MIF release signaling pathway. Pref-1 is expressed and released in Pref-1+ cells with characteristics of M2 macrophages, endothelial cells or progenitors. Pref-1 inhibits MIF release from both Pref-1+ cells and adipocytes by binding with cell membrane integrin ß1 and inhibiting the mobilization of p115, a cofactor for MIF release. High palmitic acid (PA) induces PAR2 expression in Pref-1+ cells, leading to the downregulation of Pref-1 expression and its release in an AMPK-dependent manner. When Pref-1 secretion is reduced, Pref-1+ cells and adipocytes increase MIF release and its plasma content, resulting in insulin resistance.All data are presented as mean ± SD. n = 6 each animal group. *p = 0.05 increase vs. other groups in (C, D, F and J); vs. WT to WT NC or Par2-/- to WT PD in (E); vs. NC in (O) and (P). #p = 0.05 reduction vs. other groups in (B); vs. NC in (J); vs. PD + Vehicle in (M) and (N). The n.s. represents no significance.