Fig 1: The levels of S100A16 in gastric cancer (GC) tissues and cell lines. (A) The mRNA levels of S100A2, S100A4, S100A6, S100A7, S100A8, S100A14, and S100A16 in GC tissues and their corresponding adjacent normal tissues. (B) The protein expressions of S100A16 in GC tissues and their corresponding adjacent normal tissues. The mRNA (C) and protein (D) expressions of S100A16 analyzed by real-time quantitative polymerase chain reaction (qRT-PCR) in GC cell lines (AGS, BGC-823, MKN45, MGC-803, and SGC-7901) and an immortalized normal human fallopian tube epithelial cell line FTE187 cells. All data are presented as mean ± standard error of the mean (SEM), n = 6. *p < 0.05, **p < 0.01, ***p < 0.001 versus normal tissues or FTE187.
Fig 2: S100A16 was a direct target of miR-6884-5p. (A) Schematic representation of S100A16 3'-untranslated regions (3'-UTRs) showing putative microRNA (miRNA) target site. (B) The protein levels of S100A16 were determined by Western blot in MKN45 and SGC-7901 cells transfected with miR-NC, miR-485-5p, miR-873-5p, or miR-6884-5p mimic, respectively. (C) The analysis of the relative luciferase activities of S100A16-WT and S100A16-MUT in GC cells. (D) Relative miR-6884-5p level in GC tissues and their corresponding adjacent normal tissues. (E) Relative miR-6884-5p levels analyzed by qRT-PCR in MKN45, SGC-7901, and FTE187 cells. (F) Pearson’s correlation analysis of the relative miR-6884-5p levels of and the relative S100A16 mRNA levels in GC tissues. All data are presented as mean ± SEM, n = 4. ##p < 0.01 versus miR-NC or anti-miR-NC. **p < 0.01, ***p < 0.001 versus normal tissues or FTE187.
Fig 3: Effects of S100A16 on fat metabolism and insulin resistanceC57BL/6 and S100A16Tg/+ male mice were fed a NFD or HFD from 5 to 21 weeks of age (n = 6 for C57BL/6 NFD mice, n = 5 for S100A16Tg/+ NFD mice, n = 14 for C57BL/6 HFD mice, n = 8 for S100A16Tg/+ HFD mice). (A) Body weight monitored every week. (B) Visceral fat weight measured following administration of the anesthetic Nembutal (100 mg/kg). (C) At 14 and 20 weeks, intraperitoneal glucose tolerance tests (IGTT) were performed twice. All mice were starved overnight, then injected intraperitoneally with glucose at a dose of 2 g/kg of body weight, and blood glucose levels were monitored using a handheld glucometer at 15, 30, 60, and 120 min. (D) The response in blood glucose following an intraperitoneal injection of insulin (0.3 unit/kg of body weight). At 15 and 21 weeks, insulin tolerance tests (ITT) were performed twice. All mice were starved for 12 h, then injected with a bolus of insulin (0.3 unit/kg of body weight), and blood glucose levels were monitored using the handheld glucometer at 15, 30, 60, and 120 min, and blood glucose concentrations at different time points were expressed as a percentage of the fasting blood glucose concentrations. (E) At the end of the study, adipose and liver tissues were removed immediately, and S100A16 protein levels were determined by a Western blotting with an S100A16-specific antibody. (F) Relative expression of S100A16 based on grayscale analysis with a-tubulin as a control. (G) Images of visceral fat cells and liver cells. Adipose and liver tissues were removed, immediately fixed with formalin and embedded in paraffin, and subjected to hematoxylin and eosin (HE) staining (bar = 20 µm).
Fig 4: Effects of S100A16 and 11ß-HSD1 on preadipocyte differentiation(A) Analysis of S100A16 and 11ß-HSD1 protein levels by Western blotting in protein extracts collected at 0, 4, 8, and 10 days after induction of differentiation with a-tubulin as a control. (B) Quantification of S100A16 and 11ß-HSD1 expression described in (A) based on grayscale analysis. (C) Western blotting of S100A16 protein levels in various transfectants. (D) Quantification of S100A16 expression described in (C) based on grayscale analysis. (E) Western blotting analysis of 11ß-HSD1 protein levels in various transfectants. (F) Quantification of 11ß-HSD1 expression described in (E) based on grayscale analysis. (G) Induction of differentiation of 3T3-L1 transfectants overexpressing 11ß-HSD1 or S100A16 into adipocytes followed by fixing and staining with oil red O at different time points (0, 4, and 10 days). And induction of differentiation of 3T3-L1 transfectants in which 11ß-HSD1 or S100A16 were down-regulated into adipocytes followed by fixing and staining with Oil Red O at different time points (0, 4, and 10 days). Photographs were taken using a light microscope at 200× magnification (LEICA). P =0.05 compared to controls. (H) Quantitative determination of triglyceride accumulation in cells. The lipid levels were determined using a Triglyceride GPO-POD assay kit. Results were expressed as the mean±SD for n = 3. P = 0.05 when compared with the corresponding results in the control cell lines.
Fig 5: Effect of S100A16 on 11ß-HSD1 activity(A) Mouse embryonic fibroblasts (MEFs) from C57BL/6, S100A16Tg/Tg and S100A16KO/+ mice were treated with cycloheximide (CHX) at 20 µM for different times (0, 12, or 24 h) and cell lysates were subjected to immunoblotting using anti-11ß-HSD1 antibody. (B) Quantification of 11ß-HSD1 expression described in (A) based on grayscale analysis. *P=0.05 compared to controls.
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