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: Nature Communications

Article Title: NF-κB inducing kinase is a therapeutic target for systemic lupus erythematosus

doi: 10.1038/s41467-017-02672-0

Figure Lengend Snippet: NIK is required for TWEAK-induced gene regulation. a Western blot analysis of p52 processing in mouse and human renal proximal tubulo-interstitial cells (RPTEC) stimulated with TWEAK in the presence or absence of NIK SMI1 (human 1 μM, mouse 3 μM) at 4, 8, and 24 h. b – d Differential gene expression (RNASeq) in murine RPTEC stimulated with TWEAK (100 ng/mL) in the presence or absence of 3 μM NIK SMI1 for 6 or 24 h. b Heat map of TWEAK inducible genes (FC > 2, p < 0.05) inhibited by NIK SMI1 (FC < −1.5, p < 0.05) ( c ) Plot of TWEAK inducible and NIK regulated genes at 6 and 24 h. TWEAK inducible—NIK regulated genes, black; TWEAK inducible—not NIK regulated, red; unaffected by TWEAK, but regulated by NIK, blue. d RNASeq heat map of TWEAK inducible—NIK-dependent genes that are specifically relevant to lupus. e – g Confirmation by qRT-PCR of select genes identified by RNASeq. qRT-PCR data represented as mean ± standard deviation of three biological replicates run in experimental duplicate, and normalized to expression of HPRT. Unstimulated + DMSO (gray bar), TWEAK (100 ng/mL) + DMSO (white bar), and TWEAK (100 ng/mL) + 3 μM NIK SMI1 (black bar). h Heat map, TWEAK inducible—NIK-dependent genes significantly elevated in kidneys of IFNα-accelerated NZB/W F1 mice with proteinuric disease (FC > 2, p < 0.05). Statistics: e – g one-way ANOVA with Tukey’s test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001; ns not significant

Article Snippet: Equivalent stimulations were also done with human RPTEC cells (LONZA) stimulated with rhTWEAK and 1 μM NIK SMI1 (~human IC 90 ) for 6 and 24 h for RT-PCR measurements.

Techniques: Western Blot, Gene Expression, Quantitative RT-PCR, Standard Deviation, Expressing