Journal: Scientific Reports

Article Title: Three dimensional modeling of biologically relevant fluid shear stress in human renal tubule cells mimics in vivo transcriptional profiles

doi: 10.1038/s41598-021-93570-5

Figure Lengend Snippet: Analysis of human proximal tubular albumin reuptake function. Albumin uptake by human proximal tubular epithelial cells after 15 min incubation at 37 °C with 50 µg mL-1 of FITC-albumin (green) added to the channel under static conditions ( A ) and fluid shear stress conditions ( B ). Mean Fluorescence Intensity (MFI) of RPTEC/TERT1 cells 24 h after static conditions versus fluid shear stress conditions showing a significant increase in FITC-Albumin fluorescence signal ( C ) (***p ≤ 0.001). Transcriptome profiling provides insight to differentially expressed genes corresponding to endocytosis process.

Article Snippet: Human immortalized hRPTECs (RPTEC/TERT1, ATCC CRL-4031) and were cultured and maintained in hTERT Immortalized RPTEC Growth Kit (ATCC ACS-4007), supplemented with Geneticin (Gibco, 10131035), in phenol red free DMEM/F-12 medium (Gibco, 11039021) according to the vendor's instructions.

Techniques: Incubation, Shear, Fluorescence

Journal: Scientific Reports

Article Title: Three dimensional modeling of biologically relevant fluid shear stress in human renal tubule cells mimics in vivo transcriptional profiles

doi: 10.1038/s41598-021-93570-5

Figure Lengend Snippet: Fluorescent transporter substrate Calcein-AM and CMFDA amasses in RPTEC/TERT1 cells under static conditions and dissipates with the application of FSS. Figure ( B ) and ( D ) sample fluorescence images demonstrate decreased in Calcein-AM and CMFDA accumulation in cells after 24 h of FSS treatment prior to staining compared to static channels ( A and C ) (Bar = 100 µm). ( E ) Quantitative analysis of Mean Fluorescence Intensity (MFI) of Calcein-AM showed a significant decrease after cells were treated with 24 h of FSS versus static environment (yellow filled bars). Indicating the increased efflux activity after being kept under fluidic conditions (Bar = 100 µm; n = 4, ***p ≤ 0.001).

Article Snippet: Human immortalized hRPTECs (RPTEC/TERT1, ATCC CRL-4031) and were cultured and maintained in hTERT Immortalized RPTEC Growth Kit (ATCC ACS-4007), supplemented with Geneticin (Gibco, 10131035), in phenol red free DMEM/F-12 medium (Gibco, 11039021) according to the vendor's instructions.

Techniques: Fluorescence, Staining, Activity Assay

Journal: International Journal of Biological Sciences

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure

doi: 10.7150/ijbs.38841

Figure Lengend Snippet: CtBP2 formed transcriptional complexes with p300 and c-Jun or c-FOS. (A) In vivo pull-down of the Flag-c-Jun- and c-FOS-associated complexes. RPTEC/TERT1 OAT3 cells were transfected with pCDNA3-2×Flag, pCDNA3-2×Flag-c-FOS or pCDNA3-2×Flag-c-Jun. After incubation for another 48 h, cells were subjected to immunoprecipitation analysis. The purified protein complexes were loaded onto SDS-PAGE gels, and protein bands were visualized using sliver staining. The IgG, c-Jun, c-FOS and CtBP2 bands are indicated. (B) Verification of the association of CtBP2, p300 and c-Jun or c-FOS in vivo . Protein samples used for mass spectrometry were applied to western blotting analyses to verify the association of CtBP2, p300 and c-Jun or c-FOS. (C) CtBP2 could not interact directly with c-Jun or c-FOS. The RPTEC/TERT1 OAT3 cells were transfected with the following combinations of plasmids: pCDNA3-6×Myc+pCDNA3-2×Flag-c-Jun; pCDNA3-6×Myc+pCDNA3-2×Flag-c-FOS; pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-c-Jun; and pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-c-FOS. After 48 h, cells were subjected to immunoprecipitation analysis using anti-Flag agarose and anti-Myc agarose, respectively. The pull-down products were used to determine protein interactions by probing with the antibodies indicated in the figures. (D) CtBP2 directly interacts with p300. The RPTEC/TERT1 OAT3 cells were transfected with the following combinations of plasmids: pCDNA3-6×Myc+pCDNA3-2×Flag; pCDNA3-6×Myc+pCDNA3-2×Flag-p300; pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag; and pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-p300. After 48 h, cells were subjected to immunoprecipitation analysis using anti-Flag agarose and anti-Myc agarose, respectively. The pull-down products were used to determine protein interactions by probing with the antibodies indicated in the figures. (E) p300 directly interacted with c-Jun or c-FOS. The RPTEC/TERT1 OAT3 cells were transfected with the following combinations of plasmids: pCDNA3-6×Myc+pCDNA3-2×Flag-c-Jun; pCDNA3-6×Myc+pCDNA3-2×Flag-c-FOS; pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-c-Jun; and pCDNA3-6×Myc-CtBP2+pCDNA3-2×Flag-c-FOS. After 48 h, cells were subjected to immunoprecipitation analysis using anti-Flag agarose and anti-Myc agarose, respectively. The pull-down products were used to determine protein interactions by probing with the antibodies indicated in the figures.

Article Snippet: A normal human renal cell line, RPTEC/TERT1 OAT3, was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA, #CRL-4031-OAT3).

Techniques: In Vivo, Transfection, Incubation, Immunoprecipitation, Purification, SDS Page, Staining, Mass Spectrometry, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure

doi: 10.7150/ijbs.38841

Figure Lengend Snippet: Knockdown of CtBP2 caused the repression of TGFB1 . (A) The CtBP2 mRNA level. The RPTEC/TERT1 OAT3 cells were transfected with two independent siRNAs of CtBP2 . After incubation for another 48 h, cells were further treated with or without 50 ng/mL IL-1β for 2 h, followed by RNA isolation and qRT-PCR analysis to measure CtBP2 mRNA levels. *** P < 0.001. (B) The TGFB1 mRNA level. RNA samples used in (A) were applied to qRT-PCR analysis to measure TGFB1 mRNA levels. ** P < 0.01 and *** P < 0.001. (C) Knockdown of CtBP2 inhibited TGF-β signaling. Cells used in (A) were subjected to protein isolation and western blotting to examine the protein levels of CtBP2, TGF-β, pSmad2, Smad2 and GAPDH. (D) Treatments with CtBP inhibitors could not change the mRNA level of CtBP2 . The RPTEC/TERT1 OAT3 cells were treated with 5 μM MTOB or NSC95397 for 4 h. Cells were then further treated with or without 50 ng/mL IL-1β for 2 h. The resulting cells were subjected to RNA isolation and qRT-PCR analysis to measure CtBP2 mRNA levels. (E) Treatments with CtBP inhibitors significantly repressed TGFB1 mRNA levels. RNA samples used in (D) were applied to qRT-PCR analysis to measure TGFB1 mRNA levels. *** P < 0.001. (F) Treatments with CtBP inhibitors inhibited TGF-β signaling. Cells used in (D) were subjected to protein isolation and western blotting to examine the protein levels of CtBP2, TGF-β, pSmad2, Smad2 and GAPDH.

Article Snippet: A normal human renal cell line, RPTEC/TERT1 OAT3, was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA, #CRL-4031-OAT3).

Techniques: Knockdown, Transfection, Incubation, Isolation, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure

doi: 10.7150/ijbs.38841

Figure Lengend Snippet: Knockdown or blockage of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . (A) Knockdown of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . The RPTEC/TERT1 OAT3 cells were transfected with two independent siRNAs of CtBP2 . After incubation for another 48 h, cells were further treated with or without 50 ng/mL IL-1β for 2 h, followed by ChIP assays using anti-CtBP2, anti-p300, anti-c-Jun and anti-c-FOS to determine their occupancies in the promoter of TGFB1 . ** P < 0.01 and *** P < 0.001. (B) Blockage of CtBP2 decreased the occupancies of p300 and AP-1 in the promoter of TGFB1 . The human RPTEC/TERT1 OAT3 cells were treated with 5 μM MTOB or NSC95397 for 4 h. Cells were then further treated with or without 50 ng/mL IL-1β for 2 h. The resulting cells were subjected to ChIP assays using anti-CtBP2, anti-p300, anti-c-Jun and anti-c-FOS to determine their occupancies in the promoter of TGFB1 . ** P < 0.01 and *** P < 0.001.

Article Snippet: A normal human renal cell line, RPTEC/TERT1 OAT3, was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA, #CRL-4031-OAT3).

Techniques: Knockdown, Transfection, Incubation

Journal: JCI Insight

Article Title: The TGF- β /HDAC7 axis suppresses TCA cycle metabolism in renal cancer

doi: 10.1172/jci.insight.148438

Figure Lengend Snippet: ( A ) Immunoblot analysis of ACO2 and SUCLG1 in patient-matched normal kidney (N) and tumor (T) ( n = 6). ( B ) Western blot analysis of ACO2 and SUCLG1 in a panel of RCC cell lines relative to RPTEC primary renal proximal tubule epithelial cells. The band intensities for ACO2 and SUCLG1 protein levels were quantified with reference to actin control bands using ImageJ program (NIH). ( C ) Protein expression of ACO2 and SUCLG1 across cancer subtypes from the CPTAC cohorts. ( D ) Heatmap representing expression patterns of TCA cycle enzymes in normal kidney ( n = 72), VHL mutant RCC ( n = 224), and VHL WT RCC ( n = 225). Data were extracted from TCGA KIRC data set. The heatmap shows the log 10 -transformed TPM values for each gene. ( E ) Heatmap representing expression patterns of TCA cycle enzymes using the Illumina Human HT-12 v4 bead array in the 3-patient groups (normal, n = 9; primary, n = 9; and metastasis, n = 26). Colors in the heatmap represent log-transformed quantile normalized expression values. ( F ) Relative mRNA expression of TCA cycle enzymes in a separate cohort of patient-matched samples. Transcript levels were normalized to those of TBP ( n = 4–6). Asterisks indicate significant differences compared with normal kidney (* P < 0.05, ** P < 0.01, 1-way ANOVA with Tukey’s multiple-comparison test).

Article Snippet: Primary RPTEC were acquired from Lonza and grown in renal epithelial cell growth basal medium supplemented with 10% FBS (Atlanta Biologicals) and penicillin streptomycin (100 U/mL; Corning) in 5% CO 2 at 37°C.

Techniques: Western Blot, Expressing, Mutagenesis, Transformation Assay