Fig 2: The role of KLF3 in miR-130b-3p inhibited adipocyte differentiation. Quantitative PCR validation of (A) miR-130b-3p and (B) KLF3 expression in miR-130b-3p overexpressed preadipocytes with or without KLF3 overexpression (48 h after transfection, without differentiation induction). (C) Representative images of Oil red O (the left panel) (magnification: left, 40×, right, 400×) and Bodipy (the right panel) staining of lipid droplets in miR-130b-3p overexpressed goat differentiated adipocytes (48 h of differentiation) with or without KLF3 overexpression (magnification: 400×). (E), (F), (G) The mRNA expression level of relative genes in mimics NC+vector, miR-130b-3p+vector, and miR-130b-3p+KLF3 goat differentiated adipocytes. Data are presented as mean percentage (mean ± SD) of at least three independent experiments. *P < 0.05, **P < 0.01, ns indicates no significance.

Fig 3: Gain-of function of KLF3 promotes the lipid deposition of goat intramuscular adipocytes. (A) qPCR and WB validation of KLF3 expression in vector and KLF3 plasmid transfected preadipocytes. UXT (qPCR) or ß-actin (WB) was used as an internal control (48 h after transfection, without differentiation induction). (B) The efficiency of adipocyte differentiation was dramatically increased in KLF3 overexpression cells based on Oil red O staining (magnification: left, 40×, right, 400×). The right panel was the semi-quantitative assessment result. After photographing, the cells were destined in 1 mL 100% isopropanol for 15 min and the Oil red signal was quantified by measuring the absorbance at 490 nm. (C) Representative images of Bodipy staining of lipid droplets in goat differentiated adipocytes (48 h of differentiation) with or without KLF3 overexpression (magnification: 400×). (D) Triglyceride content detection in vector and KLF3 expression plasmid transfected differentiated adipocytes. (E), (F), (G) The mRNA expression level of relative genes in vector and KLF3 expression plasmid transfected differentiated adipocytes were detected by qPCR. UXT was used as an internal control. Data are presented as mean percentage (mean ± SD) of at least three independent experiments. *P < 0.05, **P < 0.01.

Fig 4: Both miR-130b-3p and miR-130b-5p directly regulate KLF3 expression. (A) TargetScan, miRDB, and starBase were used to predict the potential targets of bovine miR-130b-3p and miR-130b-5p, the lower panel was the binding sequence. (B) TargetScan, miRDB, and starBase were used to predict miRNAs that could target goat KLF3. (C) Quantitative PCR validation of KLF3 expression in miR-130b-3p or miR-130b-5p mimics or inhibitor transfected preadipocytes. UXT was used as an internal control. Wild-type and mutant binding site of (D) miR-130b-3p and miR-130b-5p (E) in the 3'UTR of KLF3 mRNA. (F) Dual luciferase reporter assays using vectors encoding putative miR-130b-3p and miR-130b-5p target sites in the KLF3 3'UTRs for both wild-type and mutant regions. Renilla luciferase was used to normalize the firefly luciferase values. Data are presented as mean percentage (mean ± SD) of at least three independent experiments. **P < 0.01.

Fig 5: Loss-of function of KLF3 inhibits goat intramuscular adipocyte lipid deposition. (A) qPCR validation of KLF3 expression in siNC and KLF3 siRNA transfected preadipocytes. UXT was used as an internal control (48 h after transfection, without differentiation induction). (B) The efficiency of adipocyte differentiation was dramatically decreased in KLF3 downregulation cells based on Oil red O staining (magnification: left, 40×, right, 400×). The right panel was the semi-quantitative assessment result. After photographing, the cells were destined in 1 mL 100% isopropanol for 15 min and the Oil red signal was quantified by measuring the absorbance at 490 nm. (C) Representative images of Bodipy staining of lipid droplets in goat differentiated adipocytes (48 h of differentiation) with or without KLF3 interference (magnification: 400×). (D) Triglyceride content detection in siNC and KLF3 siRNA transfected differentiated adipocytes. (E), (F), (G) The mRNA expression level of relative genes in siNC and KLF3 siRNA transfected differentiated adipocytes were detected by qPCR. UXT was used as an internal control. Data are presented as mean percentage (mean ± SD) of at least three independent experiments. **P < 0.01.