Fig. 4: High glucose-associated APLN upregulation impairs metabolic homeostasis in TM4 Sertoli cell. | Nature Communications

Fig. 4: High glucose-associated APLN upregulation impairs metabolic homeostasis in TM4 Sertoli cell.

From: Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models

Fig. 4

a The expression level of Apln mRNA in TM4 cell under high glucose (HG) and insulin. Data are presented as means ± SEM. Unpaired two-tailed t test. Results of three independent experiments are shown (n = 3). b Immunofluorescence of ACTB (red) co-stained with APLN (green) in TM4 cell under HG and insulin. Scale bar, 5 μm. c Quantitative analysis of APLN immunofluorescence level in (b). Box-and-whisker plots denote the maximum (top whisker), 75th (top edge of box), 25th (bottom edge of box) and minimum (bottom whisker) percentiles, and the median (line in box). Unpaired two-tailed t test. n = 100 cells examined over 3 independent experiments. d Immunofluorescence of ACTB (red) co-stained with HIF1A (green) in TM4 cell under HG. Scale bar, 5 μm. e Genome browser view of the HIF1A density in the Apln range in negative control (NC) and HG treated TM4 cell. f PCA analysis of metabolomic data in NC and APLN group. Four biological replicates were performed for each group. g Heatmap showing all differential metabolites in NC and APLN group. h The metabolic level of NAD+, carnitine and glutathione and Palmitelaidic Acid in NC and APLN group. unpaired two-tailed t test between sample groups. n = 4 biologically independent samples. Each box represents the median and the 25 and 75% quartiles, and the whiskers indicate 1.5 times of the interquartile range. i, Immunoblots of TJP1 and GJA1 in TM4 treated APLN and by combination of three metabolites in different concentrations. j Immunoblots of TJP1 and GJA1 in TM4 treated APLN and three different concentrations (200, 500, 1000 μM) of each metabolites separately. k Immunoblots of indicated proteins in stable knockdown APJ cell treated APLN or HG. l Immunoblots of indicated proteins in TM4 treated APLN or HG and rescued by ML221. m Immunoblots of indicated proteins in TM4 treated APLN or HG and rescued by F13A. n Immunoblots of AMPKα1 and p-AMPKα1 in TM4 treated APLN or different concentrations of Palmitelaidic Acid.

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