Journal: BioMed Research International

Article Title: MicroRNAs as Salivary Markers for Periodontal Diseases: A New Diagnostic Approach?

doi: 10.1155/2016/1027525

Figure Lengend Snippet: Comparison of methods from current investigations regarding miRNAs in periodontal disease.

Article Snippet: Naqvi et al. 2014 [ ] , Human THP-1-differentiated macrophages , miRNeasy kit (Qiagen) , NanoString nCounter miRNA assay (NanoString Technologies) , Quantitative real-time PCR EvaGreen Master Mix (Biotium) , — , RNU6B , Student's t -test (two-tailed) , miR-29b miR-32 miR-146a miR-891.

Techniques: Comparison, RNA Extraction, Biomarker Discovery, Control, In Vitro, Microarray, Isolation, TaqMan microRNA Assay, SYBR Green Assay, Labeling, Real-time Polymerase Chain Reaction, Virus, Quantitative RT-PCR, In Vivo, Expressing, Mann-Whitney U-Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Representative immunofluorescence images and quantifications showing that 3‐month‐old Hnf1a pKO mice have increased number of KI67 + (red) acinar cell nuclei co‐staining with DAPI (blue) and Amylase (green). Arrows point to KI67 + acinar cells in Hnf1a pKO mouse. Acinar proliferation is represented as the average of the KI67 + /Amylase + cell ratio. Quantifications were performed on 3 random fields from 3 Pdx1 Cre and 3 Hnf1a pKO mice. P ‐values are from two‐tailed Student's t ‐test. Representative H&E stainings of pancreata from Kras G12D and Hnf1a pKO ; Kras G12D mice. B–D Kras G12D and Hnf1a pKO ; Kras G12D mice have normal morphology at 7 days. E–J At 21 days, Hnf1a pKO ; Kras G12D mice show acinar‐to‐ductal metaplasia (dashed encircled areas) and regions with desmoplastic reaction (asterisk), which are not observed in Kras G12D mice (E, H). K–P At 8 weeks, Kras G12D pancreas show occasional abnormal ductal structures (dashed encircled areas in N, which is a magnification of squared dotted box in K) and Hnf1a pKO ; Kras G12D mice (L, M, O, P) present mucinous tubular complexes (black arrows), and more advanced PanINs with luminal budding (open arrows) including foci of spindle cell proliferation (asterisks) and incipient infiltrative growth (black dashed box area in O). Data information: Black dashed boxes in (E, F, K, L and O) indicate magnified areas in (H, G, N, M and P) respectively. Scale bars indicate 200 μm (A), 100 μm (C, E, F, K, L), 50 μm (O), and 20 μm (B, D, G, H–J, M, N, P).

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Breeding strategy to generate Hnf1a aKO and Ptf1a Cre ;Hnf1a +/+ control mice using Ptf1a Cre and Hnf1a LoxP alleles. B Ptf1a Cre deletes HNF1A efficiently in acinar cells but to a lesser extent in islets of Langerhans. HNF1A IHC and hematoxylin staining in pancreas of control and Hnf1a aKO mice. HNF1A is expressed in acinar and islet cells, but not in ductal cells in normal pancreas (left). HNF1A expression is depleted in acinar cells but largely not in islets in Hnf1a aKO pancreas (right). The squared dotted boxes (top) indicate magnified areas (bottom). Arrows point at ducts, arrow head at HNF1A‐positive acinar cell, and open arrow head at HNF1A positive islet cell. The dotted encircled areas indicate islets of Langerhans. Scale bar (top) 300 μm (bottom) 50 μm. C H&E stainings in pancreas of control (left) and Hnf1 aKO mice (right) showing unaltered pancreatic morphology. Scale bar 300 μm. D Expression of acinar differentiation genes in pancreas from Hnf1a aKO and controls, depicted as box plots with median values and IQR of TPM values. Whiskers extend to highest and lowest data points within 1.5× IQR outside box limits. P ‐values were determined by two‐tailed Student's t ‐test and n = 3 replicates per condition. E GSEA showing increased expression of oncogenic pathways in Hnf1a aKO pancreas. F Western blots (top) and quantifications (bottom) showing increased phospho‐p42 levels in Hnf1a aKO pancreas. Quantification of signal intensities of phospho‐p44/p42 normalized to total‐p44/p42 levels. Data are shown as dots with mean and error bars ± SD. P ‐values were determined by two‐tailed Student's t ‐test. G Distribution of pancreatic HNF1A binding sites in annotated genomic regions. H Venn diagrams illustrating that HNF1A‐bound regions are enriched in regions of active promoters and enhancers. P ‐values and odds ratios were calculated by Fisher's exact test. I Enrichment of known HNF1 motifs in the top 500 most significant HNF1A‐bound ChIP‐seq regions and percentage of regions containing each motif. The “union” is the percentage of regions with at least one motif sequence occurrence. Enrichment P ‐values are calculated using the one‐tailed binomial test. J Genome browser track for Fn1 and Timp1 genes showing upregulated expression in Hnf1a aKO and Kdm6a pKO pancreas, and absence of HNF1A or KDM6A binding in adjacent regions. Plots show TPM values normalized to Hprt with mean and error bars ± SD. P ‐values were determined by two‐tailed Student's t ‐test.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Staining, Expressing, Two Tailed Test, Western Blot, Binding Assay, ChIP-sequencing, Sequencing, One-tailed Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Fold change (FC) in transcripts in Hnf1a aKO versus control pancreas, plotted against significance (−Log 10 q; genes significant at q < 0.05 are shown as colored dots above the horizontal line). B GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1a aKO pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage‐enriched genes were obtained from Muraro et al . C Top functional annotations for differentially expressed genes in Hnf1a aKO pancreas. D GSEA revealed that Hnf1a aKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT, MAPK, KRAS, PI3K‐AKT. E HNF1A promotes transcriptional activation of direct target genes. Left: HNF1A‐bound genes were enriched among genes that showed downregulation in Hnf1a mutants, but not among upregulated genes. P ‐values and odds ratios (O.R.) calculated by Fisher's exact test. Right: Venn diagrams showing overlap of HNF1A‐bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Functional Assay, Activation Assay, Mutagenesis

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Human orthologs of genes that were up‐ and downregulated in Hnf1a aKO pancreas were also up‐ and downregulated in human pancreas with low versus high HNF1A expression (lowest versus highest expression deciles, respectively). A random list of 717 genes controlled for similar expression levels was used for comparison. Violin plots include median and interquartile ranges. Dots are average values for each gene. Kruskal–Wallis P < 0.0001. B GSEA demonstrates that down‐ or upregulated genes in Hnf1a aKO mice (downward or upward arrows) showed down‐ or upregulation, respectively, in gene lists ranked by differential expression in non‐classical versus classical PDAC molecular subtypes from the TCGA‐PAAD study (Cancer Genome Atlas Research Network, Electronic Address Aadhe, Cancer Genome Atlas Research N, ). All enrichments had GSEA FDR q ‐values < 0.01. C Analysis of HNF1A function in 121 high‐purity cases of the ICGC‐PACA‐AU cohort identified tumors with most pronounced downregulation of direct HNF1A target genes. We performed GSEA with a gene set of 106 human orthologs of HNF1A direct targets showing downregulation in Hnf1a aKO pancreas. For each tumor sample, we performed differential expression against all other samples and used GSEA to ascertain abnormal expression of the mouse HNF1A‐dependent gene set in the tumor. Samples were ranked by the resulting normalized enrichment score (NES) and classified as either HNF1A LoF samples (purple, NES < 0; P < 0.05), or Control 1 (beige, NES < 0; P > 0.05) and Control 2 (gray, NES > 0). HNF1A LoF samples were predominantly non‐classical tumors (Collisson et al , ; Moffitt et al , ; Bailey et al , ). Putative loss‐of‐function KDM6A mutations ( KDM6A LoF mutants) were found in 19% of HNF1A LoF tumors versus 2% of all others (Fisher's P = 0.005). KDM6A mutations were considered functional if classified as “high” functional impact in ICGC (small ≤ 200‐bp deletions/insertions, single base substitutions), or as likely loss‐of‐function structural variants in Bailey et al , all of which were frame‐shift mutations. Other KDM6A mutations were classified as unknown. Heatmaps show Z ‐score‐normalized expression of deregulated genes in Hnf1a aKO pancreas. We confirmed that 85% of 106 downregulated and 60% of genes of 146 upregulated human orthologs showed differential expression across the 3 HNF1A profiles ( q < 0.05, SAM multiclass analysis). D HNF1A mRNA levels differed in HNF1A LoF and control groups (Kruskal–Wallis, P < 0.01), despite considerable variability and overlap between groups. E KDM6A mRNA levels were downregulated in HNF1A LoF tumors (Kruskal–Wallis, P < 0.001). Data information: Box plots in (D and E) show HNF1A and KDM6A expression in HNF1A LoF tumors ( n = 26) and Control 1 ( n = 39) and Control 2 ( n = 57) tumors. The horizontal central line marks the median. Box limits indicate the first and third quartiles, and whiskers extend to highest and lowest data points within 1.5× interquartile range outside box limits.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Expressing, Functional Assay

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Consensus clustered Z‐score‐normalized gene expression heatmaps of high‐purity TCGA‐PAAD and ICGC‐PACA‐AU human PDAC samples. Clustering was performed with non‐negative matrix factorization based on expression of significantly down‐ and upregulated genes in Hnf1a aKO pancreas. This revealed a cluster (HNF1A cluster 3) with concordant up‐ and downregulation of genes in Hnf1a aKO pancreas, which predominantly matched non‐classical PDAC molecular subtypes (quasimesenchymal, basal, squamous‐like, pink in top tracks), as opposed to classical PDAC subtypes (green in top tracks). Multiclass SAM differentially expressed genes ( q < 0.05) between HNF1A clusters are shown. Genes were hierarchically clustered using complete linkage with one minus Pearson correlation metrics. Along the right side of the heatmaps are green and red indicators of down‐ and upregulated genes in Hnf1a aKO pancreas, respectively. B TP63 expression was increased in HNF1A LoF tumors compared to control tumors. RSEM normalized count data are shown as box plots with interquartile range, median, and whiskers. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5× IQR outside box limits. HNF1A LoF ( n = 26), Control 1 ( n = 39), and Control 2 ( n = 57) tumors (P, Kruskal–Wallis). C, D Expression of HNF1A and KDM6A , showing downregulation in non‐classical PDAC subtypes (P, Kruskal–Wallis). Dots are RSEM normalized values presented with mean ± SD. Collisson subtypes: Quasimesenchymal (QM, n = 34) and Classical (CL, n = 54). Moffitt subtypes: Basal (BA, n = 65) and Classical (CL, n = 85). Bailey subtypes: Squamous‐like (SQ‐like, n = 31) and Pancreatic Progenitor (PP, n = 53). E, F HNF1A levels are not lower in high histological grade PDAC (E), while KDM6A levels are (F). To determine whether histological grade of human PDAC was associated with expression levels of HNF1A (E) or KDM6A (F) proteins, we evaluated contingency tables of tumor grades versus staining intensities of each case in tissue microarray (TMA) IHC. Tumor grades were scored as either moderately differentiated (G2) or poorly differentiated/high grade (G3), and staining intensities were expressed as an Immuno Reactivity Score (IRS) reflecting either No, Weak, Moderate, or Strong staining intensities (see material and methods for details). Numbers of cases and percentages (in brackets) out of total cases are indicated for each tumor grade and staining intensity. The Chi‐squared test was used to determine the probability of a significant relationship. Chi‐square and P ‐values are shown. N = 217 patients for HNF1A and N = 208 patients for KDM6A.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Expressing, Staining, Microarray

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Efficient deletion of Kdm6a in the pancreatic epithelium at E15.5. KDM6A (red) is ubiquitously expressed in all pancreatic cells. CDH1 (green) marks epithelial cells. Upon deletion, KDM6A staining is lost specifically in CDH1‐expressing epithelial cells but not in mesenchymal cells or in the stomach epithelium (white arrow heads). Scale bar indicates 100 μm. B Kdm6a mutant mice show normal fasting and fed glycemia. The horizontal stroked line indicates blood glucose levels at 250 mg/dl as a reference. C–H The pancreas of Kdm6a pKO mice were histologically normal until 8 weeks of age. At 8 weeks of age, some signs of acinar cell attrition and fat replacement could be observed, as shown in this representative image. Scale bars: 250 μm (10× magnification), 50 μm (40× magnification). I Representative picture (left) showing increased number of KI67 (red) amylase‐expressing acinar cells (green) in Kdm6a pKO pancreas. Scale bar, 250 μm. Quantifications (right) were performed on three pancreatic sections separated by at least 100 μm from 4 control and 4 Kdm6a pKO mice. Acinar cell proliferation was represented as the average of the KI67 + /Amylase + cell ratio ± SD. P ‐values were determined by two‐tailed Student's t‐ test. J GSEA plots showing enrichment of Oncostatin M and “TNFA signaling via NFKB” gene sets among genes upregulated in Kdm6a pKO pancreas. K Western blots (top) and quantifications (bottom) showing increased phospho‐p44/p42 levels in Kdm6a pKO pancreas. Quantification of signal intensities of phospho‐p44/p42 normalized to total‐p44/p42 levels. Data are shown as dots with mean and error bars ± SD. P ‐values were determined by two‐tailed Student's t ‐test. L Most significantly deranged REACTOME pathways in both KDM6A‐ and HNF1A‐deficient pancreas (see also ). M Kdm6a pKO down‐ and upregulated gene sets showed concordant deregulation in KDM6A LoF mutant tumors versus classical PDAC (based on Bailey et al 's signature) (Bailey et al , ). N Tumors with KDM6A‐deficient phenotypes showed decreased KDM6A mRNA. We created a gene set of human orthologs of Kdm6a pKO downregulated genes, and for each high‐purity tumor sample in the ICGC‐PACA‐AU, we used GSEA to test for enrichment of this gene set in gene lists that were rank‐ordered by differential expression in the individual sample versus all other samples. Samples with NES < 0 and P ‐value < 0.05 were considered as having KDM6A LoF phenotypes and were compared against all other samples. Z ‐score‐normalized count data are shown as box plots with IQR, median, and whiskers. Whiskers extend to highest and lowest data points within 1.5× IQR outside box limits. P ‐values were determined by two‐tailed Student's t ‐test. O Gene sets that showed up‐ or downregulation in non‐classical human PDAC showed concordant enrichment in up‐ or downregulated genes in Kdm6a pKO versus control pancreas. GSEA NES and FDR q‐values are shown. P Genomic distribution of KDM6A binding sites in mouse pancreas. Q, R Top: ChIP‐seq and RNA‐seq tracks in control and Kdm6a pKO pancreas, in two loci harboring downregulated genes ( Kif12 , Gprc5c ) in Kdm6a pKO pancreas. Bottom: ChIP‐qPCR validations for regions highlighted in green (R1, R2, R3), showing that Kdm6a mutants have increased H3K27me3 and decreased H3K27ac in most regions. H3K4me1 was also decreased in mutants in distal sites. Error bars show SD, and P ‐values were determined by two‐tailed Student's t ‐test, n = 3. S KDM6A‐bound regions are enriched in active pancreas promoters and enhancers. P ‐values are calculated by Fisher's exact test. T, U Genome Browser examples (top) of HNF1A and KDM6A binding to genes known as negative regulators of EMT: Gstp1 (T) and Deptor in (U) that are downregulated in Hnf1a aKO and Kdm6a pKO pancreas (bottom). Plots show TPM values normalized to Hprt with mean and error bars ± SD. N = 4 per condition and P ‐values were determined by two‐tailed Student's t ‐test.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Staining, Expressing, Mutagenesis, Two Tailed Test, Western Blot, Binding Assay, ChIP-sequencing, RNA Sequencing Assay

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Motif analysis in functional KDM6A‐bound regions, showing top ten de novo motifs ranked by P ‐value determined by HOMER software. B Co‐binding analysis in functional KDM6A‐bound enhancer and promoter regions revealed that HNF1A was the most enriched co‐bound TF among three other acinar cell TFs. Binding regions of TAL1 in a non‐pancreatic cell type and random binding sites were used as negative controls. P ‐values were determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background. C The most downregulated genes in Kdm6a pKO pancreas are shown ranked by q‐value and are almost invariably bound by HNF1A and downregulated in Hnf1a aKO pancreas, or known to be direct HNF1A‐dependent target genes from other studies (red and purple, respectively). D, E GSEA analysis on the Hnf1a aKO and Kdm6a pKO ranked‐ordered gene lists versus their reciprocal up‐ or downregulated gene sets, demonstrated that KDM6A and HNF1A regulate similar genes. F Expression changes in Hnf1a aKO and Kdm6a pKO pancreas, showing that genes bound by KDM6A and downregulated in Kdm6a pKO pancreas (red dots) were generally downregulated in Hnf1a aKO pancreas. G HNF1A and KDM6A co‐occupy the same regions in Pah , which is downregulated in Hnf1a and Kdm6a knock‐out pancreas. H Genes that were co‐bound by KDM6A and HNF1A showed greatest downregulation in Kdm6a pKO pancreas, compared with KDM6A‐bound genes that were not bound by HNF1A. Box plots show median and IQR of Log 2 TPM fold‐changes and whiskers extend to highest and lowest data points within 1.5× IQR outside box limits. P ‐values were determined by two‐tailed Student's t ‐tests and n = 4 replicates per condition.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Functional Assay, Software, Binding Assay, Expressing, Knock-Out, Two Tailed Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A–D Left: Genome Browser examples of loci co‐bound by HNF1A and KDM6A, showing loss of KDM6A binding in HNF1A‐deficient pancreas (region highlighted in green) and decreased RNA levels in HNF1A‐deficient pancreas. Right: ChIP‐qPCRs showing loss of KDM6A and HNF1A binding in highlighted regions in left and qPCRs show downregulation of target genes in Hnf1a ‐KO pancreas. Error bars show SD, and P ‐values were determined by two‐tailed Student's t ‐test, n = 4 for ChIP‐qPCRs and n = 3 for qPCRs.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Binding Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A HNF1A binding to chromatin is unaffected in Kdm6a pKO pancreas. Scatterplot showing unchanged HNF1A binding in Kdm6a pKO versus control pancreas (e.g., see also Appendix Fig S6A–D ). B Western blot showing KDM6A depletion in two clones from Kdm6a ‐KO acinar cell lines. C–F qPCR in Kdm6a ‐KO acinar cell lines (KO1 and KO2) shows reduced expression of HNF1A bound genes, while ChIP‐qPCRs for HNF1A show that its binding to those genes is unchanged when KDM6A is depleted. Selected genes and HNF1A binding regions were from Appendix Fig S6A–D . qPCR data are relative mRNA expression of indicated genes versus Hprt . ChIP‐qPCR values indicate fold enrichment relative to control region. Error bars show ± SD, and P ‐values were determined by two‐tailed Student's t ‐test. G GSEA analysis on ranked‐ordered gene list from Kras G12D ;Kdm6a pKO data from Andricovich et al versus gene sets from up‐ or downregulated genes in Hnf1a aKO and Kdm6a pKO pancreas demonstrates that KDM6A and HNF1A regulate similar genes in normal and Kras G12D ‐transformed pancreas. H GSEA, comparing rank‐ordered expression data from Kras G12D ;Kdm6a pKO , Hnf1a aKO , and Kdm6a pKO mice with gene sets from Andricovich et al , shows that most of the pathways that are enriched in Kdm6a ‐deficient pancreatic cancer are dependent on HNF1A and KDM6A function in the non‐tumoral pancreas.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Binding Assay, Western Blot, Clone Assay, Expressing, Two Tailed Test, Transformation Assay

Journal: The EMBO Journal

Article Title: HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

doi: 10.15252/embj.2019102808

Figure Lengend Snippet: A Co‐immunoprecipitation of endogenous HNF1A and KDM6A followed by Western blot demonstrated that HNF1A is in the same complex as KDM6A. B Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a −/− pancreas. C Differential binding analysis of KDM6A in Hnf1a −/− versus wild‐type pancreas. Pink dots below zero (1,873 sites) show regions with reduced KDM6A binding, and pink dots above zero (118 sites) are regions with increased binding at FDR < 0.05. D, E Regions that show reduced KDM6A binding in Hnf1a −/− chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P ‐values in (D) were calculated with two‐tailed Mann–Whitney U‐test and in (E) with Fisher's exact test. F KDM6A binding is markedly reduced in HNF1A‐ and KDM6A‐co‐bound regions in Hnf1a −/− pancreas, but not in other KDM6A‐bound regions. G Genes that loose KDM6A binding in Hnf1a ‐mutant pancreas are predominantly downregulated in Hnf1a aKO pancreas and are direct HNF1A target genes (red dots). H Summary model depicting that HNF1A recruits KDM6A to genomic binding sites, activating an acinar differentiation program that indirectly suppresses core oncogenic pathways. Defective HNF1A or KDM6A function results in failure of this shared program, with increased activity of pathways that, in the presence of KRAS mutations, promote high‐grade non‐classical PDAC with sarcomatoid features.

Article Snippet: Endogenous peroxidase and protein blocking was performed with 3% H 2 O 2 diluted in PBS for 10 min and with 1% BSA, 10% normal goat serum (Abcam, Cambridge, UK), and 0.1% Triton X‐100 (Merck KGaA, Darmstadt, Germany) for 60 min. Anti‐HNF1A and anti‐KDM6A stainings were performed at a dilution of 1:250 (Anti‐HNF1A, Abcam ab204306, Cambridge, UK), 1:200 (Anti‐HNF1A, Cell Signaling Technology, 89670, Leiden, The Netherlands), and 1:100 (Anti‐UTX, Cell Signaling Technology 33510S, Denver, USA), respectively.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Two Tailed Test, MANN-WHITNEY, Mutagenesis, Activity Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Loss of ARG1 and AGMAT enhances liver tumor formation (A) Immunoblots of arginine-to-polyamine-converting enzymes (ARG1 and AGMAT) and polyamine metabolism enzymes (ODC, SRM, SMS, SAT1, PAOX, and SMOX) in Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control (same samples were used as in Figure 1 E). n = 4 (Ctrl), n = 8 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO tumor tissues. n = 6. (C) Relative 3 H-putrescine uptake into Ctrl liver and L-dKO tumor tissues. n = 8. (D) Immunohistochemistry of Ctrl and L-dKO liver tissues stained for ARG1 or AGMAT. NT, adjacent non-tumor tissue; T, tumor. (E) Representative images of livers from L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. (F) Number of macroscopic tumors per liver of L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 9–10. (G) Arginine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. ∗ p < 0.05, ∗∗ p < 0.01. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by unpaired t test (B and C) and one-way ANOVA (F and G).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Western Blot, Control, Immunohistochemistry, Staining, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Loss of ARG1 and AGMAT promote tumorgenicity by sustaining high levels of arginine, related to Figure 2 (A) Polyamine species in L-dKO tumors relative to Ctrl liver tissues (log 2 ratio). n = 5 (Ctrl), n = 6 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice fed with arginine-modified diets. n = 3–9. (C) Immunohistochemistry of Ctrl and L-dKO liver tissues from 12- and 16-week-old mice stained for ARG1 or AGMAT proteins, respectively. NT, adjacent non-tumor tissue; T, tumor. (D) Immunoblots of ARG1 and AGMAT in paired L-dKO non-tumor (NT) and tumor (T) tissues from mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. AKT serves as loading control. n = 2 (AAV-Ctrl), n = 3 (AAV-ARG1), and n = 3 (AAV-AGMAT). (E) Liver-to-body-weight ratio of Ctrl and L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. (F) Total polyamine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. n.s. = not significant; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by multiple t test (A) and one-way ANOVA (B, E, and F).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Modification, Immunohistochemistry, Staining, Western Blot, Injection, Control

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1 and AGMAT expression determine metabolism and growth of liver cancer cells, related to Figure 3 (A) Immunoblots of ARG1, AGMAT, CPS1, OTC, ASS1, and ASL expression in human liver cancer cell lines. Actin serves as loading control. (B) Representative clonogenic growth assay of control, ARG1-, and/or AGMAT-expressing SNU-449 cells grown in standard, arginine-rich DMEM (i.e., 400 μM) medium. (C) Relative clonogenic growth of control, ARG1-, and/or AGMAT- expressing SNU-449 cells grown in standard, arginine-rich DMEM medium. N = 3. (D) Arginine content in plasma and TME of L-dKO mice. n = 8 (plasma), n = 6 (TME). (E) Representative clonogenic growth assay of control and ARG1/AGMAT-expressing SNU-449 cells grown in medium containing 100 μM arginine (“plasma-like”) or 20 μM arginine (“TME-like”). (F) Relative polyamine content of control, ARG1-, and/or AGMAT-expressing SNU-449 cells. N = 4. (G) Immunoblots of SNU-449 cells upon stable overexpression of ASS1-FLAG. Huh1 cells serve as control for expression of arginine synthesis enzymes. Calnexin serves as loading control. (H) Arginine content of control or ASS1-FLAG-overexpressing SNU-449 cells. (I) Representative clonogenic growth assay of control or ASS1-FLAG-overexpressing SNU-449 cells grown under arginine-restricted conditions. (J) Immunoblots of ARG1/AGMAT-expressing SNU-449 cells upon stable overexpression of ASS1 or 3xHA-ASS1. Huh1 cells serve as control for expression of arginine synthesis enzymes. Calnexin serves as loading control. (K) Arginine content of control, ASS1-, or 3xHA-ASS1-overexpressing SNU-449 ARG1/AGMAT cells. (L) Clonogenic growth assay of control, ASS1-, or 3xHA-ASS1-overexpressing SNU-449 ARG1/AGMAT cells grown under arginine-rich (400 μM) or arginine-restricted (4 μM) conditions. (M) Representative images of hepatospheres of control and ARG1/AGMAT-expressing SNU-449 cells grown in arginine-restricted medium in ultra-low attachment plates. Scale bar, 100 μm. (N) Number of hepatospheres (as in G). N = 6. (O) Immunoblot analyses of ARG1 and AGMAT in sgCtrl, sgARG1, and sgAGMAT Huh7 cells. Calnexin serves as loading control. (P) Representative clonogenic growth assay of sgCtrl, sgARG1, and sgAGMAT Huh7 cells. (Q) Relative clonogenic growth of sgCtrl, sgARG1, and sgAGMAT Huh7 cells. N = 3. (R) Clonogenic growth of ARG1/AGMAT-expressing SNU-449 cells grown in arginine-restricted medium in the presence of 400 μM of indicated metabolites. (S) Volcano plot of the −log 10 (adjusted p value) against the log 2 fold-change of the differentially expressed genes in ARG1/AGMAT-expressing compared to control SNU-449 cells. Blue and red dots indicate significantly decreased and increased gene expression, respectively. (T) Deregulated metabolic pathways (within top 25 of all deregulated pathways; see Table S2 ) in ARG1/AGMAT-expressing compared to control SNU-449 cells after PWEA (using KEGG pathways, presented by enrichment factor) of differentially expressed genes from RNA-seq. (U) mRNA levels of ASNS , PSAT1 , PSPH , GLSK , GLUT3 , and HK2 in ARG1/AGMAT-expressing SNU-449 cells grown in arginine-restricted medium with or without supplementation of excess arginine (i.e., 4 mM equal to 10× compared to standard DMEM medium) for 16 h. N = 4–8. n.s. = not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (C, F, K, and Q) and unpaired t test (D, H, N, and U).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Expressing, Western Blot, Control, Growth Assay, Over Expression, RNA Sequencing Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1/AGMAT determine metabolic gene expression via arginine (A) Immunoblots of SNU-449 cells upon stable expression of ARG1 and/or AGMAT. Actin serves as loading control. (B) Representative clonogenic growth assay of control, ARG1-, and/or AGMAT-expressing SNU-449 cells grown in arginine-restricted medium. (C) Relative clonogenic growth of control, ARG1-, and/or AGMAT- expressing SNU-449 cells. N = 6. (D) Arginine content of control, ARG1-, and/or AGMAT-expressing SNU-449 cells. N = 4. (E) PCA analysis of RNA-seq data of control and ARG1/AGMAT-expressing SNU-449 cells. (F) Heatmap of a subset of differentially expressed metabolic genes in ARG1/AGMAT-expressing compared to control SNU-449 cells (log 2 fold-change). (G) mRNA levels of ASNS , PSAT1 , PSPH , GLSK , GLUT3 , HK2 , NNMT, and AOC3 in control and ARG1/AGMAT-expressing SNU-449 cells. N = 5–7. (H) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ARG1/AGMAT-expressing SNU-449 cells. Calnexin serves as loading control. (I) Immunoblots of ASNS, PSAT, PSPH, and NNMT of Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control. n = 4 (Ctrl), n = 8 (L-dKO). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA (C and D) and unpaired t test (G).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Expressing, Western Blot, Control, Growth Assay, RNA Sequencing Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1/AGMAT-regulated ASNS enhances arginine uptake required for tumorigenicity, related to Figure 4 (A) Top ten differentially expressed genes in ARG1/AGMAT-expressing compared to control SNU-449 cells by log 2 fold-change (left) and −log 10 (adjusted p value) (right). (B) Clonogenic growth of control and ARG1/AGMAT-expressing SNU-449 cells grown in arginine-restricted medium supplemented with asparagine as indicated. (C) Clonogenic growth of ARG1/AGMAT+control or ARG1/AGMAT+ASNS-expressing SNU-449 cells grown in arginine-restricted or arginine-deficient medium. (D) mRNA levels of ATF4 and ATF4 target genes SESN2 , GPT2 , MTHFD2 , VEGFA , and SLC1A5 in control and ARG1/AGMAT-expressing SNU-449 cells grown under arginine-restricted conditions. Unpaired t test; n.s. = not significant. N = 7. (E) Representative images of livers from L-dKO mice injected with AAV-shCtrl or AAV-sh Asns . (F) Immunoblot of ASNS in non-tumor (NT) and tumor (T) tissues of L-dKO mice injected with AAV-shCtrl or AAV-sh Asns . n = 3. Calnexin serves as loading control. ∗ indicates a cross-reaction.

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Expressing, Control, Injection, Western Blot

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ASNS promotes arginine uptake in liver cancer (A) Relative 3 H-arginine uptake in control and ARG1/AGMAT-expressing SNU-449 cells with or without pre-loading with asparagine (Asn) or glutamine (Gln). N = 5–6. (B) Immunoblots of ARG1/AGMAT-expressing SNU-449 cells upon stable expression of ASNS or control. Calnexin serves as loading control. (C) Relative 3 H-arginine uptake in control and ASNS-expressing SNU-449 ARG1/AGMAT-expressing cells. N = 5. (D) Representative clonogenic growth assay of control and ASNS-expressing SNU-449 ARG1/AGMAT-expressing cells grown in arginine-restricted medium. (E) mRNA levels of PSAT1 , PSPH , GLSK , GLUT3 , HK2 , NNMT, and AOC3 in control and ASNS-expressing SNU-449 ARG1/AGMAT-expressing cells. N = 6–8. (F) Immunoblots of ASNS, PSAT, PSPH, and NNMT from two independent experiments of control and ASNS-expressing SNU-449 ARG1/AGMAT-expressing cells. Calnexin serves as loading control. (G) mRNA levels of Asns in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-shCtrl or AAV-sh Asns . n = 6–7. (H) Number of macroscopic tumors per liver in L-dKO mice injected with AAV-shCtrl or AAV-sh Asns . n = 7. (I) Arginine content in L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-shCtrl or AAV-sh Asns . n = 4–6. n.s. = not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by unpaired t test (A, C, E, G, and H) and one-way ANOVA (I).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Control, Expressing, Western Blot, Growth Assay, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: RBM39 requires arginine binding to transcriptionally control metabolic gene expression and tumorigenicity, related to Figure 6 (A) mRNA levels of ASNS , PSAT1 , PSPH , GLSK , GLUT3 , HK2 , NNMT , AOC3 , and RBM39 upon si RBM39 and siCtrl in SNU-449 cells. N = 5–7. (B) mRNA levels of ASNS , PSAT1 , HK2 , NNMT , and RBM39 upon stable knockdown of RBM39 (sh RBM39_1 and sh RBM39_2) and shCtrl in SNU-449 cells. N = 5–6. (C) mRNA levels of ATF4 in indisulam- or DMSO-treated SNU-449 cells. N = 6. (D) mRNA levels of ASNS , PSAT1 , PSPH , GLUT3 , and NNMT in indisulam- or DMSO-treated ARG1/AGMAT-expressing SNU-449 cells. N = 5–6. (E) mRNA levels of PSAT1 , PSPH , GLUT3 , and NNMT in indisulam- or DMSO-treated ARG1/AGMAT+ASNS-expressing SNU-449 cells. N = 4. (F) Representative clonogenic growth assay of SNU-449 shCtrl, sh RBM39_1 , and sh RBM39_2 cells grown under arginine-restricted conditions in the absence or presence of 100 μM asparagine. (G) Immunoblot of 3xHA-RBM39 expressed in ARG1/AGMAT-expressing SNU-449 cells. Calnexin serves as loading control. (H) mRNA levels of ASNS , PSAT1 , PSPH , GLSK , NNMT, HK2 , and RBM39 in control and 3xHA-RBM39-expressing SNU-449 ARG1/AGMAT cells. N = 3. (I) mRNA levels of RBM39 in indisulam- or DMSO-treated SNU-449 cells. N = 4. (J) PCA analysis of RNA-seq data of control and RBM39-depleted SNU-449 cells. (K) Volcano plot of the −log 10 (adjusted p value) against the log 2 fold-change of differentially expressed genes in RBM39-depleted compared to control SNU-449 cells. Blue and red dots indicate significantly decreased and increased gene expression, respectively. (L) Clustering of the top 2,500 differentially expressed genes in ARG1/AGMAT-expressing compared to control SNU-449 cells with the differentially expressed genes in RBM39-depleted compared to control SNU-449 cells. Values of differentially expressed genes were binarized prior to clustering. (M) Table summarizing alternative splicing events (ASEs) detected in RNA-seq of control and RBM39-depleted SNU-449 cells and control and ARG1/AGMAT-expressing SNU-449 cells after analysis with the R package NxtIRFcore. IR, intron retention by algorithm; RI, intron retention curated; SE, skipped exon; A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; AFE, alternative first exon; ALE, alternative last exon; MXE, mutually excluded exon (see also Table S4 ). (N) Read counts of TRIM27 (Tripartite motif-containing protein 27), DUSP11 (Dual specificity protein phosphatase 11), THEM4 (Thioesterase superfamily member 4), and RFC4 (Replication factor C subunit 4) from RNA-seq of control and RBM39-depleted SNU-449 cells displayed with integrated genome viewer (IGV). Regions highlighted with arrows indicate skipped exons (SE) or intron retention (IR). Blue line indicates introns, and blue boxes indicate exons. Arrow below blue line indicates gene orientation. (O) Representative endpoint PCR of TRIM27 (exon 3–8) in control and RBM39-depleted cells (as in J). (P) Read counts of ASNS , PSAT1 , GLUT3 , and HK2 from RNA-seq of control and RBM39-depleted SNU-449 cells displayed with IGV (as in N). (Q) Relative luciferase-based promoter activity of ASNS and PSAT1 in SNU-449 shCtrl, sh RBM39_1 , and sh RBM39_2 cells grown under arginine-restricted conditions. N = 4–6. (R) Relative luciferase-based promoter activity of ASNS and PSAT1 in control and ARG1/AGMAT-expressing SNU-449 cells grown under arginine-restricted conditions. N = 5–8. (S) Immunoblots of SNU-449 cells expressing full-length, ΔN, or ΔN-NLS cMYC RBM39(G268V)-FLAG treated with indisulam or DMSO. Calnexin serves as loading control. s.e., short exposure; l.e., long exposure. (T) mRNA levels of ASNS in SNU-449 cells expressing ΔN-NLS cMYC RBM39(G268V)-FLAG treated with indisulam for two days in arginine-restricted conditions or in arginine-repleted conditions (400 μM). N = 6. (U) Representative endpoint PCR of TRIM27 (exon 3–8) in SNU-449 cells expressing full-length, ΔN, or ΔN-NLS cMYC RBM39(G268V)-FLAG treated with indisulam. (V) Relative clonogenic growth of SNU-449 cells expressing full-length, ΔN, or ΔN-NLS cMYC RBM39(G268V)-FLAG treated with indisulam. N = 3. (W) Representative images of livers from L-dKO mice injected with AAV-shCtrl or AAV-sh Rbm39 . (X) Liver-to-body-weight ratio of L-dKO mice injected with indisulam or vehicle. n = 4 (vehicle), n = 5 (indisulam). n.s. = not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by unpaired t test (A–E, H, I, Q, R, T, and X) and one-way ANOVA (V).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Binding Assay, Control, Expressing, Knockdown, Growth Assay, Western Blot, RNA Sequencing Assay, Alternative Splicing, Luciferase, Activity Assay, Injection

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1, AGMAT, arginine, and RBM39 in human HCC patients (A) Schematic representation of arginine and polyamine metabolism in HCC patients. Boxes below enzymes indicate changes in mRNA (left box) and protein (right box) levels in human HCC tumors (T) compared to paired non-tumor (NT) biopsies, respectively. Color coding according to level of log 2 fold-change as indicated. “?” indicates unknown identity. Tumor aggressiveness is indicated by Edmondson-Steiner grade low (Edm. low, grade I and II) and high (Edm. high, grade III and IV). n = 73 (Edm. low) and n = 49 (Edm. high) for mRNA; n = 30 (Edm. low) and n = 21 (Edm. high) for protein. (B) Immunoblots of ARG1, AGMAT, RBM39, and ASNS in paired non-tumor (NT) and tumor (T) tissues of five HCC patients. Calnexin serves as loading control. (C) Tissue microarray for ARG1 and AGMAT. ARG1, normal liver n = 58, HCC n = 160; AGMAT, normal liver n = 49, HCC n = 142. (D) Representative IHC of ARG1 and AGMAT of an HCC patient (from C). Non-tumor, NT; tumor, T. (E) Kaplan-Meier survival estimate curve for The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) patients ranked by expression of ARG1 and AGMAT . n = 89 (low), n = 109 (normal). (F) Urea cycle metabolites in tumors (T) relative to paired non-tumor (NT) liver tissues (log 2 ratio). n = 11. (G) Immunoblots of RBM39 in tumor lysate (Input) and elution after purification with leucine (Leu)- or arginine (Arg)-coupled agarose beads from three HCC patients. Calnexin serves as input and negative control. (H) Dose-response curve of 20 HCC patient-derived organoids treated with indisulam. Data are presented as the percentage of control DMSO-treated tumor organoids. (I) Model. In liver cancer cells, loss of ARG1 and AGMAT preserves arginine, which in turn binds RBM39 to promote metabolic reprogramming. Arginine-RBM39-mediated ASNS expression further enhances arginine uptake. Trsx, transcription. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 by unpaired t test (C), log rank test (E), and multiple t test (F).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Western Blot, Control, Microarray, Expressing, Purification, Negative Control, Derivative Assay

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: ARG1 and AGMAT are decreased and arginine, RBM39, and ASNS are increased in HCC patient tumors that are sensitive to RBM39 depletion by indisulam, related to Figure 7 (A) RBM39 mRNA levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 73 (Edm. low), n = 49 (Edm. high). (B) RBM39 protein levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 30 (Edm. low), n = 21 (Edm. high). (C) ASNS mRNA levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio. n = 73 (Edm. low), n = 49 (Edm. high). (D) ASNS protein levels in liver tumor tissue (T) from HCC patients compared to adjacent non-tumor tissue (NT), displayed as log 2 ratio, if applicable. BW, black-and-white, i.e., only detected in tumor tissues. n = 3 (Edm. low), n = 8 (Edm. high). (E) Staging of ARG1 and AGMAT IHC staining in tissue micro array. (F) mRNA expression of ARG1 , AGMAT , RBM39 , and ASNS in early-stage HCC (data from Jiang et al. ). log 2 fold-change tumor (T) relative to non-tumor (NT) tissues. n = 35. (G) Kaplan-Meier survival estimate curve for TCGA-LIHC patients ranked by expression of ARG1 . n = 135 (low), n =155 (normal). (H) Kaplan-Meier survival estimate curve for TCGA-LIHC patients ranked by expression of AGMAT . n = 136 (low), n = 158 (normal). (I) Polyamine species in tumors (T) relative to paired non-tumor (NT) liver tissues (log 2 ratio). n = 11. (J) Arginine content in paired non-tumor (NT) and tumor (T) tissues of HCC patients. n = 10. (K) Total polyamine content in paired non-tumor (NT) and tumor (T) tissues of HCC patients. n = 10. (L) Volcano plot of the −log 10 (adjusted p value) against the log 2 fold-change of 600 proteins identified by MS (in minimum 2 out of 3 samples) after purification from HCC tissues by arginine (Arg)- compared to leucine (Leu)-coupled agarose beads. Red dot highlights RBM39. (M) Dose-response curve of 20 HCC patient-derived organoids treated with sorafenib. Data are presented as the percentage of control DMSO-treated tumor organoids. (N) IC 50 of indisulam- and sorafenib-treated HCC patient-derived organoids. n = 20. (O and P) Rbm39 and Asns mRNA levels in embryonic day 14 (E14), E18, and adult mouse liver as reads per kilobase of exon per million reads mapped (RPKM). Data from NBCI Gene. n.s. = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by paired t test (A–C, J, K, and N), multiple t test (F and I), and log rank test (G and H).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Immunohistochemistry, Microarray, Expressing, Purification, Derivative Assay, Control

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet:

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2α (Cell Signaling, Cat# 2103), eIF2α-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, RNA Sequencing Assay, Control, Mutagenesis, CRISPR, Plasmid Preparation, shRNA, Software

Journal: Nature cell biology

Article Title: Medial HOXA genes demarcate haematopoietic stem cell fate during human development

doi: 10.1038/ncb3354

Figure Lengend Snippet: (A, B) Microarray analysis of HOXA gene expression in CD34 + CD38 −/lo CD90 + GPI-80 + cells and their progeny (Mean values are shown, left, n=3 samples, GEO database GSE54316, and right, n=3 samples (CD34+CD38-CD90+) or 2 (CD34+CD38-CD90- and CD34+CD38-) GSE34974. (C) Schematic showing the strategy for lentiviral shRNA knockdown of HOXA5 or HOXA7 in FL-HSPCs. (D) Knockdown is confirmed using q-RT-PCR 1 week post-infection (mean +/- SD shown from n=3 different FL samples). (E) Representative FACS plots 30 days after HOXA5 or HOXA7 knockdown. (F) Quantification of HSPC subsets in empty-vector (CTR) and shRNA infected cells (shHOXA5 or shHOXA7) after 5, 14 and 30 days in culture (mean and SEM, n=6 independent experiments per condition for day 14 and n=3 for day 5 and 30). Statistical significance was assessed using Wilcoxon Signed Rank test. (G) Schematic showing the transplantation strategy with HOXA5 or HOXA7 knockdown FL-HSPCs. (H) Representative FACS plots from mouse BM 10 weeks post-transplantation assessing human CD45 + cells and multi-lineage engraftment (CD19 and CD3 for B-and T-lymphoid, and CD66 and CD33 for myeloid). (I) Quantification of human engraftment (n=9 mice per condition from 3 independent experiments Individual values and mean are shown.) Statistical significance was assessed using the Wilcoxon Rank Sum test (J) RNA-sequencing of HOXA7 knockdown FL-HSPCs at day 5 post-infection. Number of genes up- or down-regulated in sh HOXA7 FL-HSPCs are shown. Genes dysregulated both in HOXA7 knockdown FL-HSPCs (RNA-seq 1.8-fold change, n=4 independent experiments, p-value < 0.05) and in EB-OP9-HSPCs compared to FL-HSPCs (microarray, 2-fold change, p-value < 0.05) are shown in blue pattern overlay. (K) Examples of HSC factors downregulated in HOXA7 knockdown FL-HSPCs and (L) differentiation associated genes upregulated in HOXA7 knockdown FL-HSPCs. Mean shown for n=4 independent specimens, values used to generate graphs can be found in and GEO database GSE76685). See for Statistics source data for 4D, F and I.

Article Snippet: Human HOXA5, HOXA7 and HOXA9 were cloned from human FL full-length cDNA, into either the constitutive pFUGW lentiviral vector, (Addgene plasmid # 14883, from David Baltimore), downstream and in frame with the GFP sequence with the synthetic addition a P2A sequence between the 2 ORFs, or the inducible lentiviral overexpression system pNL-EGFP/TREPittdU3, (Addgene plasmid # 18659, from Jakob Reiser), between the sites BamHI and NheI. pNL-TREpitt vectors were co-transduced with the constitutive pNL-EF1α-rTTA-M2 lentiviral vector to provide in trans the Tet transactivator.

Techniques: Microarray, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Transplantation Assay, RNA Sequencing Assay

Journal: Nature cell biology

Article Title: Medial HOXA genes demarcate haematopoietic stem cell fate during human development

doi: 10.1038/ncb3354

Figure Lengend Snippet: (A) Schematic showing the strategy for constitutive lentiviral overexpression of HOXA5 or HOXA7 in FUGW vectors in FL-HSPCs. (B) Representative FACS plots of FUGW empty vector, HOXA5- or HOXA7- overexpressing FL-HSPCs. (C, D) Expansion of total FL cells (C) or HSPCs (D) transduced with HOXA5- or HOXA7- overexpression vectors or empty vector control (CTR), (mean and SEM values from n=3 independent experiments; statistical significance was assessed using the paired Student’s t -test. (E) q-RT-PCR confirming overexpression in transduced HSPCs sorted 1 week post-infection (n=1 experiment with 2 pooled donors). (F) CFU-Cs from 2000 HSPCs sorted after day 10 of infection with vectors overexpressing HOXA5 or HOXA7 or FUGW empty vector control (mean and SD values shown from n=4 transductions from 2 independent experiments, p-values shown correspond to CTR vs. OE-HOXA7). (G) Schematic showing the strategy for lentiviral overexpression of HOXA5 and/or HOXA7 and/or HOXA9 in FUGW vectors in EB CD34 + cells. (H) Representative examples of FACS plots of EB CD34 + cells overexpressing HOXA5 or HOXA7 or a combination of HOXA5 , HOXA7 and HOXA9 . Un-transduced FL is shown as a control. (I) Quantification of CD34 + CD38 −/lo CD45 + haematopoietic cells from (H), mean from n=4 independent experiments for CTR and n=3 for HOXA5/7/9, HOXA5, and HOXA7 at days 0 and 24, and n=2 at all other time points. J) Representative FACS plots and (K) quantification of human CD45 + cells in the BM of NSG mice 12 weeks post-transplantation. Multi-lineage engraftment is assessed by CD19 and CD3 (B-and T-lymphoid) and CD66 and CD33 (myeloid) (mean from n=5 mice per condition (except for FL n=4) from two independent experiments). (L). Q-RT-PCR for HOXA7 from transduced EB-OP9-HSPCs 2 weeks post-infection from one representative experiment. (M) Graphs representing RNA-seq of EB-OP9 cells overexpressing HOXA7 for genes regulated by HOXA7 in FL-HSPCs (one representative experiment, GEO database GSE76685). See for statistics source data in D, E, F, I and K.

Article Snippet: Human HOXA5, HOXA7 and HOXA9 were cloned from human FL full-length cDNA, into either the constitutive pFUGW lentiviral vector, (Addgene plasmid # 14883, from David Baltimore), downstream and in frame with the GFP sequence with the synthetic addition a P2A sequence between the 2 ORFs, or the inducible lentiviral overexpression system pNL-EGFP/TREPittdU3, (Addgene plasmid # 18659, from Jakob Reiser), between the sites BamHI and NheI. pNL-TREpitt vectors were co-transduced with the constitutive pNL-EF1α-rTTA-M2 lentiviral vector to provide in trans the Tet transactivator.

Techniques: Over Expression, Plasmid Preparation, Transduction, Reverse Transcription Polymerase Chain Reaction, Infection, Transplantation Assay, RNA Sequencing Assay