Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: (A) Endogenous PBRM1 was immunoprecipitated (IP) from human embryonic kidney cells (HEK293, left) and RCC cells (ACHN, right), and co-purified proteins were analyzed by LCMS/MS. Shown are identified coactivator (CoA) components and transcription factors. Circle size indicates abundance of protein in the interactome. A minimum Mascot ion score of 25 and peptide rank 1 were used for automatically accepting all peptide MS/MS spectra. The most enriched functional group was “ATPase chromatin remodeling complex” (p value Bonferroni-corrected 1.42 × 10 −27 ). The STRING database system was used to construct the protein-protein interaction network with a parameter STRONG score >0.4. Detection, quantification, Gene Ontology (GO), and STRING data for these and other proteins are in . Heatmap shows quantification versus IP with immunoglobulin G (IgG) isotype control. (B) Heatmap summary of the data shown in (A). (C) Bi-directional IP-western blots in 293T and three RCC cell lines confirmed the interaction between PBRM1 and PAX8. The RCC cell lines used were ACHN, SKRC45, and SKRC29.

Article Snippet: The cleaned supernatant was then incubated with Mouse anti-PAX8 (SCBT, sc-81353) and normal mouse IgG ((SCBT, sc-2025) overnight at 4° C. Protein A agarose/Salmon Sperm DNA (Millipore, Cat # 16-157) was added and incubated for another 1 hour at room temperature.

Techniques: Immunoprecipitation, Purification, Tandem Mass Spectroscopy, Functional Assay, Construct, Western Blot

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: (A) Endogenous PAX8 was IP from PBRM1-deficient RCC cells (SKRC-45 with deletion of a PBRM1 allele), and coregulator interactions were analyzed by LCMS/MS. A minimum Mascot ion score of 25 and peptide rank 1 were used for automatically accepting all peptide MS/MS spectra. By GO analyses, the most enriched protein functional groups were the NURD and CBX corepressor(CoR) complexes (p value Bonferroni corrected 3.47 × 10 −29 ). STRING was used to construct the protein-protein interaction network with a STRONG score >0.4. Circle size indicates abundance of protein in the interactome. Detection, quantification data, GO, and STRING analyses data are in . Heatmap shows quantification versus IP with IgG isotype control. (B) Endogenous PAX8 was IP from PBRM1-deficient RCC cells (ACHN with mutation of a PBRM1 allele) and coregulator interactions analyzed by LCMS/MS. Analyses as described for (A).

Article Snippet: The cleaned supernatant was then incubated with Mouse anti-PAX8 (SCBT, sc-81353) and normal mouse IgG ((SCBT, sc-2025) overnight at 4° C. Protein A agarose/Salmon Sperm DNA (Millipore, Cat # 16-157) was added and incubated for another 1 hour at room temperature.

Techniques: Tandem Mass Spectroscopy, Functional Assay, Construct, Mutagenesis

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: (A) The analysis approach. (B) Copy numbers of genes for components of the PBAF CoA and for druggable CoR components recruited by PAX8. Gistic thresholded copy-number data from TCGA (n = 342). (C) The copy-number alterations (predominantly deletions) and inactivating mutations to genes for PBAF CoA components impact their expression accordingly. Gene expression in normal kidney (NKid) and RCCs stratified by recurrent deletions and mutations of CoA genes (RNA sequencing [RNA-seq] TCGA, NKid cortex n = 72, RCC n = 342). Median ± interquartile range (IQR). *p < 0.001, two-sided Mann-Whitney test. (D) The copy-number alterations to genes for druggable CoR components (predominantly gains/amplifications) impact their expression accordingly. Gene expression in NKid and RCCs stratified by recurrent gains/amplifications of CoR genes (RNA-seq TCGA, NKid cortex n = 72, RCC n = 342). Median ± IQR. *p < 0.001, two-sided Mann-Whitney test.

Article Snippet: The cleaned supernatant was then incubated with Mouse anti-PAX8 (SCBT, sc-81353) and normal mouse IgG ((SCBT, sc-2025) overnight at 4° C. Protein A agarose/Salmon Sperm DNA (Millipore, Cat # 16-157) was added and incubated for another 1 hour at room temperature.

Techniques: Expressing, RNA Sequencing Assay, MANN-WHITNEY

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: (A) The proximal tubule core transcription factor circuit . (B) PAX8 localizes at regulatory regions of GATA3 , LHX1 , and WT1 . Chromatin IP (ChIP)-qRT-PCR analyses using α-PAX8 and IgG isotype control; primers amplified proximal promoter regions. Mean + SD for three biological replicates. Two-sided unpaired t test. (C) Selective loss in RCC (CAKI1)of H3K27Ac but not H3K4me3 at key PAX8 transcription factor target-genes ( GATA3 , LHX1 , and WT1 ; red boxes). NKid, normal kidney. H3K27Ac and H3K4me3 public ChIP-seq data (Encode and E-MTAB-7812; ). (D) GATA3 / LHX1 / WT1 are least activated in RCCs with biallelic PBRM1 inactivation (Del+Mut, deletion and mutation). NKid n = 72, RCC with PBRM1 Del n = 256, RCC with PBRM1 Del+Mut n = 180 (TCGA, RNA-seq). Boxplot = median ± IQR, whiskers = range. *p < 0.001, #p < 0.05, two-sided Mann-Whitney test. (E) GATA3 / LHX1 / WT1 CpG methylation is greatest in RCCs with PBRM1 Del+Mut. CpG numbers at each gene: PAX2 , 49; PAX8 , 16; GATA3 , 28; LHX1 , 31; and WT1 , 46. NKid n = 160, RCC with PBRM1 Del n = 74, RCC with PBRM1 Del+Mut n = 64 (TCGA, 450K Illumina array). (F) >1,000 proximal tubule genes (kidney epithelial genes) are consistently suppressed in RCCs versus NKid. Genes identified from kidney development and normal tissues gene expression databases. Two-sided unpaired t test for average expression/gene NKid versus RCCs. (G) H3K27Ac but not H3K4me3 loss at repressed proximal tubule genes (F); ChIP-seq per (C). Two-sided unpaired t test, average ChIP-seq values/gene.

Article Snippet: The cleaned supernatant was then incubated with Mouse anti-PAX8 (SCBT, sc-81353) and normal mouse IgG ((SCBT, sc-2025) overnight at 4° C. Protein A agarose/Salmon Sperm DNA (Millipore, Cat # 16-157) was added and incubated for another 1 hour at room temperature.

Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Amplification, ChIP-sequencing, Mutagenesis, RNA Sequencing Assay, MANN-WHITNEY, CpG Methylation Assay, Expressing

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: (A) The experimental approach. (B) Western blot and IP-western blot analyses of empty vector versus FLAG-PBRM1 transfected RCC (ACHN) cells. PAX8 versus IgG control IP was performed in lysates from empty vector versus PBRM1-FLAG transfected cells 48 h after transfection. (C) Heatmaps to indicate amounts of coregulators in the PAX8 interactome in empty vector versus FLAG-PBRM1 transfected cells. PAX8 was IP and proteins analyzed by LCMS/MS. Analyses were 48 h after transfection. A minimum Mascot ion score of 25 and peptide rank 1 were used for automatically accepting all peptide MS/MS spectra. STRING was used to construct the protein-protein interaction network with a STRONG score >0.4. Circle size indicates abundance of protein in the interactome. Detection, quantification, GO, and STRING analyses data are in . (D) Relative abundances of CoA and CoR complexes with empty vector versus PBRM1 transfection. The individual proteins constituting CoA and CoR are listed in (A). Median ± IQR. Values analyzed are tabulated in .

Article Snippet: The cleaned supernatant was then incubated with Mouse anti-PAX8 (SCBT, sc-81353) and normal mouse IgG ((SCBT, sc-2025) overnight at 4° C. Protein A agarose/Salmon Sperm DNA (Millipore, Cat # 16-157) was added and incubated for another 1 hour at room temperature.

Techniques: Western Blot, Plasmid Preparation, Transfection, Tandem Mass Spectroscopy, Construct

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: (A) The experimental approach. (B) Heatmaps compare amounts of coregulators in the endogenous PAX8 interactome in vehicle versus Dec-treated RCC cells (SKRC-45). LCMS/MS analyses of proteins pulled down by PAX8 IP. Vehicle or Dec 0.5 μM added on days 1 and 2 and analyses done on day 3. A minimum Mascot ion score of 25 and peptide rank 1 were used for automatically accepting all peptide MS/MS spectra. STRING was used to construct the protein-protein interaction network with a STRONG score >0.4. Circle size indicates abundance of protein in the interactome. Detection, quantification data, GO, and STRING analyses data are in . (C) Relative abundances of specific CoA (green) and CoR (red) complexes with vehicle versus Dec treatment. The individual proteins constituting each complex are listed in (B). Median ± IQR. Values analyzed are tabulated in . (D) Western blot and IP-western blot analysis of the PAX8 protein hub in vehicle versus Dec-treated RCC cells. PAX8 versus IgG control IP in lysates from vehicle, Dec, and camptothecin 10 μM (CP, as conventional chemotherapy control) treated RCC (SKRC-45) cells.

Article Snippet: The cleaned supernatant was then incubated with Mouse anti-PAX8 (SCBT, sc-81353) and normal mouse IgG ((SCBT, sc-2025) overnight at 4° C. Protein A agarose/Salmon Sperm DNA (Millipore, Cat # 16-157) was added and incubated for another 1 hour at room temperature.

Techniques: Tandem Mass Spectroscopy, Construct, Western Blot

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The cleaned supernatant was then incubated with Mouse anti-PAX8 (SCBT, sc-81353) and normal mouse IgG ((SCBT, sc-2025) overnight at 4° C. Protein A agarose/Salmon Sperm DNA (Millipore, Cat # 16-157) was added and incubated for another 1 hour at room temperature.

Techniques: Recombinant, Protease Inhibitor, DNA Purification, Software

Journal: International Journal of Molecular Sciences

Article Title: Resveratrol Alleviates the Inhibitory Effect of Tunicamycin-Induced Endoplasmic Reticulum Stress on Expression of Genes Involved in Thyroid Hormone Synthesis in FRTL-5 Thyrocytes

doi: 10.3390/ijms22094373

Figure Lengend Snippet: Effect of resveratrol (RSV), tunicamycin (TM) and TM and RSV on expression of genes involved in transcriptional regulation of thyroid hormone synthesizing genes in FRTL-5 cells. After reaching confluence, cells were treated with either 0.1% DMSO (control), 10 µM RSV, 0.1 µg/mL TM or 10 µM RSV and 0.1 µg/mL TM in 6H medium for 48 h. ( A , B ) Relative mRNA levels ( A ) and protein levels ( B ) of TTF-1, TTF-2, PAX-8 and TSHR are expressed as fold of control. Representative immunoblots for TTF-1, TTF-2, PAX-8 and TSHR including immunoblots for β-Actin and Vinculin as internal controls are shown on the right. Data from qPCR are means and SD calculated from three replicates for the same treatment of three independent experiments and were subjected to 2-factorial (T = treatment, E = experiment) ANOVA. Data from immunoblotting are means and SD calculated from one replicate for the same treatment of three independent experiments and were subjected to 1-factorial ANOVA. Bars without the same letters ( a,b,c,d ) differ ( p < 0.05).

Article Snippet: The blots were incubated with the following primary antibodies at 4 °C overnight: rabbit anti-HSPA5 (1:5000), mouse anti-DDIT3 (1:2000), mouse anti-NIS against deglycosylated NIS (1:500) (all from Thermo Fisher Scientific, Darmstadt, Germany), rabbit anti-NIS against both glycosylated and deglycosylated NIS (1:2000) (kindly provided from Prof. Nancy Carrasco, Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA), mouse anti-TTF-1 (1:500), mouse anti-TTF-2 (1:500), mouse anti-PAX-8 (1:500), rabbit anti-TSHR (1:1000) (all from Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-TG against both glycosylated and deglycosylated TG (1:5000; Abcam, Cambridge, UK), and either mouse anti-β-actin (1:20,000; Abcam) or rabbit anti-vinculin (1:10,000; Thermo Fisher Scientific, Darmstadt, Germany) as reference proteins for normalization at RT for 2 h. After washing, the blots were incubated with the following secondary antibodies—anti-rabbit-IgG (1:10,000; Sigma-Aldrich, Taufkirchen, Germany) or anti-mouse IgG antibody (1:10,000; Santa Cruz Biotechnology, Heidelberg, Germany) at RT for 2 h. Blots were developed using ECL Plus (GE Healthcare, München, Germany) and band intensities were evaluated densitometrically as described recently [ ].

Techniques: Expressing, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney

doi: 10.1681/ASN.2019090962

Figure Lengend Snippet: Expression of Pax2 and Pax8 in newborn and adult kidneys. Immunostaining for Pax2 (red [A–D]) and Pax8 (red [E and F]) is shown with either cadherin1 (Cdh1), LTL, or DBA in green. Newborn kidneys show strong Pax2 in the nephrogenic zone ([A] arrows) and in collecting ducts (arrowheads), whereas Pax8 is in all proximal, distal, and collecting tubules (E). The adult cortex is negative for Pax2 in LTL-positive proximal tubules (B) but remains Pax2-positive in LTL-negative collecting ducts ([B] arrow), whereas LTL-positive epithelia exhibit strong Pax8 expression (F). The outer medulla shows strong Pax2 in DBA-positive collecting ducts ([C] arrows), whereas Pax8 is strong in DBA-positive and DBA-negative epithelial cells of the outer medulla (G). Cells in the renal papilla show strong Pax2-positive (D) and Pax8-positive (H) nuclei in all epithelial nuclei.

Article Snippet: 17 , 18 Rabbit IgG (011–000–003) was from Jackson ImmunoResearch (West Grove, PA), anti– β -actin (A-1978) was from Sigma (St. Louis, MO), anti-H3K4me3 (ab8580) and anti-Pol II CTD (ab5408) were from Abcam (Cambridge, UK), anti–Ash2-like (Ash2L, A300–107A) and anti–retinoblastoma-binding protein 5 (RBBP5, A300–109A) were from Bethyl Laboratories (Montgomery, TX); and anti-Pax8 antibodies (sc-81353 mouse and sc-16279 goat) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Immunostaining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney

doi: 10.1681/ASN.2019090962

Figure Lengend Snippet: Gross phenotypes of Pax2/8 double mutant kidneys. (A) Histology of adult kidneys with the indicated genotypes after tamoxifen administration shows no major structural abnormalities of pathology. Slight dilation of IMCDs is seen in the Pax2/8 KOs (right lower panel), which may reflect increased urine output. (B) Water consumption as measured in single-housed metabolic cages of mice with no Pax2/8 deletion (Cre−) or deletions of either Pax2 (P2), Pax8 (P8), or both genes (P28). A significant increase in water consumption was observed in Pax2/8 double KOs (*P<0.001; n=6 per genotype). (C) Urine outputs as collected from metabolic cages over 24-hour periods shows significant increase in urine excretion only in Pax2/8 double KOs (*P<0.001; n=6 per genotype). (D) Osmolality was measured in urines from Pax2/8 double KOs (Cre+) and controls (Cre−). (E) Immunostaining for Pax2 or Pax8 (green) in kidneys from Pax2fl/fl;Pax8fl/fl mice with or without RosaCreEr after tamoxifen administration. (F) Quantitative RT-PCR for Pax2 or Pax8 mRNA from three total kidney RNAs per cohort after tamoxifen treatment of control mice (Cre−), Pax2, Pax8, or Pax2/8 double KOs.

Article Snippet: 17 , 18 Rabbit IgG (011–000–003) was from Jackson ImmunoResearch (West Grove, PA), anti– β -actin (A-1978) was from Sigma (St. Louis, MO), anti-H3K4me3 (ab8580) and anti-Pol II CTD (ab5408) were from Abcam (Cambridge, UK), anti–Ash2-like (Ash2L, A300–107A) and anti–retinoblastoma-binding protein 5 (RBBP5, A300–109A) were from Bethyl Laboratories (Montgomery, TX); and anti-Pax8 antibodies (sc-81353 mouse and sc-16279 goat) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Mutagenesis, Immunostaining, Quantitative RT-PCR

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney

doi: 10.1681/ASN.2019090962

Figure Lengend Snippet: Regulation of UTA1 and UTA2 in Pax2/8 mutants. Adult kidney sections from control mice or mice carrying floxed alleles of either Pax2, Pax8, or Pax2 and Pax8 after tamoxifen treatment were immunostained with antibodies against Slc14a2 (red) or the brush border protein villin (green). Low-power images on the left show staining in the outer medulla (UTA2) of controls and single mutants, but not in the Pax2/8 double KOs. Villin marks the proximal tubule rich cortex. Higher-powered images through the inner medulla are shown on the right, with little detectable apical UTA1 in the Pax2/8 double KOs. Representative sections from three independently derived kidneys are shown for each genotype.

Article Snippet: 17 , 18 Rabbit IgG (011–000–003) was from Jackson ImmunoResearch (West Grove, PA), anti– β -actin (A-1978) was from Sigma (St. Louis, MO), anti-H3K4me3 (ab8580) and anti-Pol II CTD (ab5408) were from Abcam (Cambridge, UK), anti–Ash2-like (Ash2L, A300–107A) and anti–retinoblastoma-binding protein 5 (RBBP5, A300–109A) were from Bethyl Laboratories (Montgomery, TX); and anti-Pax8 antibodies (sc-81353 mouse and sc-16279 goat) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Staining, Derivative Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney

doi: 10.1681/ASN.2019090962

Figure Lengend Snippet: Mechanism of Pax8-activated gene expression. Human HEK293 cells carrying an integrated Pax reporter gene (A) driven by Pax Responsive Sequences (PRS) were used to characterize Pax8-mediated transcription activation. (B) Cells were treated with negative control short hairpin RNA(shN) or PTIP KO short hairpin RNA (shPTIP) and transfected with Pax8 or an shPTIP-resistant rescue plasmid (lane 4). Note that PTIP KOs fail to activate the GFP reporter gene in the presence of Pax8. (C–H) Chromatin from cells transfected as in (B) was immunoprecipitated with antibodies against the indicated proteins. Quantitative PCR was done with primers against the PRS sequence to determine protein occupancy. Note that Pax8 binds regardless of PTIP KOs. However, Pax8 recruits PTIP and components of the Mll3/4 complex (Ash2L and Rbbp5) to imprint high levels of H3K4me2 and H3K4me3. All ChIP assays were performed in triplicate, with error bars representing one SD from the mean.

Article Snippet: 17 , 18 Rabbit IgG (011–000–003) was from Jackson ImmunoResearch (West Grove, PA), anti– β -actin (A-1978) was from Sigma (St. Louis, MO), anti-H3K4me3 (ab8580) and anti-Pol II CTD (ab5408) were from Abcam (Cambridge, UK), anti–Ash2-like (Ash2L, A300–107A) and anti–retinoblastoma-binding protein 5 (RBBP5, A300–109A) were from Bethyl Laboratories (Montgomery, TX); and anti-Pax8 antibodies (sc-81353 mouse and sc-16279 goat) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Activation Assay, Negative Control, shRNA, Transfection, Plasmid Preparation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney

doi: 10.1681/ASN.2019090962

Figure Lengend Snippet: Pax8-dependent osmoregulation of Slc14a2. (A) Protein lysates from IMCD cells cultured with 500 mM NaCl were isolated at various times after salt addition and blotted for Pax8 and Pax2. (B) Expression of UTs was measured post salt addition. (C) As in (B), AQP mRNAs were measured in response to high-salt addition in IMCD cells. (D) A schematic of exons 1 and 2 of the Slc14a2 gene on mouse chromosome 18 is shown with the position of primer pairs used for ChIP indicated. (E and F) IMCD cells cultured under 300 or 500 mM NaCl show increase in Pax8 protein and increased UTA1 and UTA3 expression (*P<0.01). (G–M) ChIP experiments using chromatin form IMCD cells cultured with 300 or 500 mM NaCl and immunoprecipitated with the indicated antibodies. Note binding of Pax8 near primer pairs 4 and 6, the recruitment of PTIP, Ash2L, and Rbbp5, and the increase in H3K4me2 and RNA Pol II. ChIP PCR reactions were run in triplicate for two independent chromatin sets. Error bars are one SD from the mean.

Article Snippet: 17 , 18 Rabbit IgG (011–000–003) was from Jackson ImmunoResearch (West Grove, PA), anti– β -actin (A-1978) was from Sigma (St. Louis, MO), anti-H3K4me3 (ab8580) and anti-Pol II CTD (ab5408) were from Abcam (Cambridge, UK), anti–Ash2-like (Ash2L, A300–107A) and anti–retinoblastoma-binding protein 5 (RBBP5, A300–109A) were from Bethyl Laboratories (Montgomery, TX); and anti-Pax8 antibodies (sc-81353 mouse and sc-16279 goat) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Cell Culture, Isolation, Expressing, Immunoprecipitation, Binding Assay