Fig 2: mRNA and protein expression of RARRES2 (A,B), CCRL2 (C,D), CMKLR1 (E,F), and GPR1 (G,H) in rat uterus. mRNA expression was determined using real-time PCR and presented relative to Gapdh; the protein expression was detected via Western blot assay, each protein abundance was evaluated densitometrically and expressed as the ratio relative to the ACTB abundance. Results were mean ± SEM of eight independent determinations. Statistically significant differences (p < 0.05) between points in each graph are indicated by different letters (a < b < c < d), identical letters indicate a lack of significant differences. ACTB, β-actin. C, control. CCRL2, C-C motif chemokine receptor-like 2. CMLR1, chemerin chemokine-like receptor 1. Gapdh, glyceraldehyde-3-phosphate dehydrogenase. GPR1, G protein-coupled receptor 1. PCR, polymerase chain reaction. PCOS, polycystic ovarian syndrome. RARRES2, retinoic acid 109 receptor responder protein 2. SEM, standard error of the mean. VD3, vitamin D3.

Fig 3: Effects of CCRL2 and chemR23 removal on recognition memory of offspring of diabetic dams. Detection of rearing frequency (a) and rearing times (b) of 8-week-old offspring of a normal pregnancy and of chemerin-induced diabetic dams, chemerin-induced diabetic dams with CCRL2-knockdown, and chemerin-induced diabetic dams with ChemR23-knockdown. Measurement of crossing frequency between squares (c) and frequency of crossing the center squares (d) by 8-week old offspring. e Comparative analysis of immobility time in 8-week old offspring. *Chemerin-induced diabetic dams vs. controls, # chemerin-induced diabetic dams with ChemR23 knockdown, and chemerin-induced diabetic dams with CCRL2 knockdown vs. chemerin-induced diabetic dams. ns, not significant; * and #—P < 0.05; ** and ##—P < 0.01

Fig 4: Association between chemerin enrichment and chemokine (C-C motif) receptor-like 2 (CCRL2) in 18.5-day-old fetal mice and 7-day-old offspring. a Protein levels of chemerin, CCRL2, and ChemR23 in whole brain tissues of 18.5-day-old fetal mice and 7-day-old offspring from controls and chemerin-induced diabetic mice (tissues from one whole brain). b Immunofluorescence staining for chemerin and CCRL2 in forebrain tissue specimens of offspring from the control, chemerin-induced diabetic dams, and chemerin-induced diabetic dams with CCRL2-knockdown mice. c Detection of chemerin and CCRL2 protein expression in brain tissue by western blotting (tissues from one whole brain). d The interaction between chemerin and CCRL2 was evaluated by co-immunoprecipitation and immunoblot assays in brain tissues of 18.5-day-old fetal mice (two fetal brains). Scale bar: 50 μm

Fig 5: Genome-wide microarray analysis of BMDMs incubated with MTB Hsp16.3. Bone marrow cells were isolated from the tibias and femurs of BALB/c mice (6–8 weeks old) and incubated with 20 ng/ml GM-CSF for 7 days. Then, BMDMs were treated with 100 ng/ml MTB Hsp16.3 for 72 h, and the cells were collected. Global gene expression was analyzed by a cDNA chip array. a Heat map. b Scatterplot of gene expression. c The fold change and frequency. d Prediction of 5 target genes, including AHR, CCR2, CCRL2, CX3CR1, and Mertk. e BMDMs were treated with 100 ng/ml MTB Hsp16.3 for 72 h, cells were collected, and the mRNA expression of the indicated genes was determined by real-time PCR. f Cells were harvested in RIPA buffer containing protease and phosphatase inhibitor cocktails, and the protein expression levels of CCRL2 and CX3CR1 were determined by Western blot. Data are presented as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group (0 h group).