Figure 2. The expression of molecular clock components is up-regulated by TGF-β in proximal tubular epithelial cells.

(A, B, C) Evaluation of the expression of molecular clock genes and fibrotic markers in synchronized human proximal TEC (HPTEC) treated with TGF-β for 0, 4, 6, 8, and 12 h. (A) Relative mRNA levels of molecular clock genes, Col1a1 and Fn1. (B) Immunoblots showing the Clock, Bmal, and pSmad2 expressions. β-actin was used for normalization. (C) Relative protein expression of Bmal1 and Clock obtained by densitometry of images from C and normalized with β-actin. (D, E, F, G, H) Evaluation of the expression of Bmal1, Cry1, and Clock in synchronized HPTEC treated with TGFβ for 24 h in the presence or absence of the selective inhibitor SB505124. (D) Immunoblot depicting the expression of Clock, Bmal1, pSmad3, and Smad3. β-actin was used for normalization. (E) Bar plot represents the relative protein expression of Bmal1, Clock, Smad3, and pSmad3 obtained by densitometry of images from D and normalized with β-actin. (F) Bar plot represents the levels of relative mRNA of Bmal1 and Cry1. (G) Immunofluorescence microscopy of Bmal1; Dapi was used for the staining of the nucleus and phalloidin for the actin cytoskeleton. Scale bar: 50 μm. (H) Quantification of the fluorescence of Bmal1 calculated from 10 different fields per sample. The experiment was repeated three times, and the data are represented with bar plots as the mean of the integrated density of all fields analyzed (a total of 30 fields per condition) ± s.e.m. (I) Schematic depicting the amplification regions of the primers designed for the analysis of the expression of the mouse (blue) and human (green) pre-mRNA of the gene Arntl. (J) Relative pre-mRNA levels of Arntl in contralateral and obstructed kidneys from mice 25 d after unilateral ureteral obstruction (UUO). (K) Relative pre-mRNA levels of Arntl in synchronized human primary proximal tubular epithelial cells treated with TGF-β for 24 h. (L) Smad3 and Smad4 (positive and negative strands) sites predicted in the distal enhancer by JASPAR2018 with a relative score above 80%. (M) Representation denoting the regulatory regions located upstream of the ARNTL gene. Data obtained from the UCSC genome browser (19). The image shows the DNA sequence upstream of the human ARNTL gene and indicates distal and proximal enhancers, H3K27Ac marks and conservation among vertebrates. (N) Representation denoting the conservation among vertebrates of the R-Smads potential binding sites found in a distal enhancer close to the ARNTL gene. Data were obtained with the UCSC genome browser (19). (O) Relative mRNA levels of Col1a1, Arntl, and Smad3 in synchronized HPTEC transfected with the overexpression vector for Smad3 (Smad3–PCMV5) or the control vector (PCMV5) and treated with TGF-β for 24 h in the presence or absence of the selective inhibitor SB505124. (O, P) Immunoblots showing the Bmal1, pSmad3, and Smad3 expressions under the same conditions as (O). β-actin was used for normalization. (Q) Relative protein expression of Bmal1, pSmad3, and Smad3 obtained by densitometry of images from P and normalized with β-actin. (R) Bioluminescence assays in HPTEC transfected with the vector reporter constructions Pgl3p, hARNTLp, SmEnh-Pgl3p, SmEnh-hARNTLp, 3xCAGA, and with the overexpression vector for Smad3 treated with TGFβ for 24 h in the presence or absence of the selective inhibitor SB505124. (A, C, E, F, J, K, O, Q, R) Data information: data represent the mean ± s.e.m of (A, F, K) n = 4, (C, Q, R) n = 3, or (E, J, O) n = 6 independent experiments; each performed in triplicates. (E, F, G, H, I, J, K, L, M, N, O, P, Q, R) ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with control without treatments, *P < 0.05, **P < 0.01, ***P < 0.001 compared with cells treated with TGFβ only, ^$P < 0.05 compared with their corresponding control without Smad3 overexpression (Mann–Whitney). (A, C) ^#P < 0.05 significant throughout time (one-way ANOVA).

Source data are available for this figure.