Journal: bioRxiv

Article Title: Regulation of HDAC2-PDX1 by RNF125 defines pancreatic cancer development

doi: 10.1101/2020.01.06.896555

Figure Lengend Snippet: ( A ) Quantification of indicated genes in RNF125 Low vs RNF125 High patient samples. Statistical analysis by single-tailed Student’s t-test( n = 3 per group). ( B ) IHC staining of indicated proteins in 7-day-old KPC organoids of control (Scr), shRNF125 (#1 or #2) or RNF125RM (RING finger mutant); Scale bars = 300 μm (right), 50 μm (insets). Arrowheads indicate positive (filled) or negative (empty) nuclei stain. ( C ) Left, IHC staining with indicated proteins of KPC orthotopic tumors. Scale bars = 400 μm (upper panels) and 100 μm (insets). Right, Quantification of results from 25 fields from 2 representative mice per group. Student’s t-test was used to calculate statistical significance. *** P <0.005. ( D ) Left, IHC staining with indicated markers in RNF125 Low ( n =17) vs. RNF125 High ( n =4) human PDA samples. PDX1-negative (black arrowheads) and - positive (blue arrowheads) nuclei are marked. Scale bars = 400 μm (upper panels) and 75 μm (insets). Student’s t-test was used to calculate statistical significance. * P <0.05. ( E ) Western blot analysis with indicated antibodies in Mia PaCa-2 cells co-transfected with shRNF125 plasmid, which targets the 3’UTR region, and either wild type (WT) or RING mutant (RM) forms of RNF125, both of which lack that region. (F) RNF125 expression in four different pancreatic cancer subtypes based on . Statistical significance was calculated using one-way ANOVA. ( G ) Right, representative PDX1 staining in normal human pancreas (NP) and in PanIN and PDA specimens. Black arrowheads indicate PDX1-positive nuclei. Scale 100 μM. Left, quantification of staining (n=3; 10 fields counted for each) ( H ) Representative PDX1 staining in pancreatic samples from 3-month-old KPC mice, showing normal pancreas (N) and PanIN (P). Scale = 50 μM (PDX1).( I ) Kaplan-Meier plot comparing PDA patients with PDX1 High versus PDX1 Low tumors; two-sided log-rank P = 0.0069. * P <0.05, ** P <0.01, *** P <0.005, **** P <0.001, NS – nonsignificant.

Article Snippet: PDX1 (Addgene#114304, Addgene#114305), NR5A2 (DNASU#HsCD443475, Addgene#28093) and RFP-tagged histone H2B (Addgene#26001) expressing plasmids were obtained from Addgene or DNASU as indicated.

Techniques: Immunohistochemistry, Control, Mutagenesis, Staining, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Regulation of HDAC2-PDX1 by RNF125 defines pancreatic cancer development

doi: 10.1101/2020.01.06.896555

Figure Lengend Snippet: ( A ) Western blot (left) and qPCR (right) analyses of PDX1 and NR5A2 levels after RNF125 knockdown. qPCR results are means ± SEM in 1 of 2 experiments. ( B ) TCGA PDA dataset analysis showing combined effect of PDX1 and RNF125 levels on pancreatic cancer survival and indicating survival of RNF125 High /PDX1 High ( n = 19) compared to RNF125 Low /PDX1 Low ( n = 22) patients. Kaplan-Meier analysis and log-rank test were used; two-sided log-rank P = 0.0359. ( C ) Similar analysis for effects of NR5A2 levels on pancreatic cancer survival and indicating survival of RNF125 High /NR5A2 High patients ( n = 19) versus RNF125 Low /NR5A2 Low ( n = 22) patients. Kaplan-Meier analysis and log-rank test were used. NS – nonsignificant. ( D ) Proliferation of Mia PaCa-2 or Panc-1 lines after knockdown or overexpression of PDX1. Data are presented as means ± SEM of 3 different experiments. Statistical significance was calculated using two-way ANOVA. ( E ) RT-qPCR analysis of transcripts encoding EMT markers in Mia PaCa-2 cells 96 hours after transfection with siPDX1. Data are representative of 3 different experiments with 3 technical replicates. Statistical significance was calculated using two-way ANOVA. ( F ) RT-qPCR analysis of transcripts encoding the EMT markers Ncad or VIM in Mia PaCa-2 cells 96 hours after transfection with indicated constructs. Data are representative of 3 different experiments with 3 technical replicates. Statistical significance was calculated using two-way ANOVA.

Article Snippet: PDX1 (Addgene#114304, Addgene#114305), NR5A2 (DNASU#HsCD443475, Addgene#28093) and RFP-tagged histone H2B (Addgene#26001) expressing plasmids were obtained from Addgene or DNASU as indicated.

Techniques: Western Blot, Knockdown, Over Expression, Quantitative RT-PCR, Transfection, Construct

Journal: bioRxiv

Article Title: Regulation of HDAC2-PDX1 by RNF125 defines pancreatic cancer development

doi: 10.1101/2020.01.06.896555

Figure Lengend Snippet: ( A ) top, Representative Ki67 staining in 48-hour-old KPC organoids transduced with shContol (upper row) or shRNF125 (lower row), and overexpressing empty vector (EV; left), or PDX1 (right). Scale bar, 50μm. Bottom, number of Ki67-positive nuclei per organoid in corresponding conditions ( B ) left, Representative KPC orthotopic tumors, 3 weeks after injection of cells corresponding to those shown in (A). right, Tumor weights in corresponding conditions ( n =8 mice per group). ( C ) IHC analysis of tumors corresponding to those shown in panel B with indicated staining.

Article Snippet: PDX1 (Addgene#114304, Addgene#114305), NR5A2 (DNASU#HsCD443475, Addgene#28093) and RFP-tagged histone H2B (Addgene#26001) expressing plasmids were obtained from Addgene or DNASU as indicated.

Techniques: Staining, Transduction, Plasmid Preparation, Injection

Journal: bioRxiv

Article Title: Regulation of HDAC2-PDX1 by RNF125 defines pancreatic cancer development

doi: 10.1101/2020.01.06.896555

Figure Lengend Snippet: ( A ) left, LC-MS/MS analysis of cultured Mia PaCa-2 cells using RNF125RM as bait. right, Networks of nuclear and non-nuclear RNF125 interactors. ( B ) PDX1 transcript levels in Mia PaCa-2 cells transfected with indicated siRNAs. Results are means ± SEM in a representative experiment. ( C ) Validation of RNF125-HDAC2 interaction based on western analysis with the indicated antibodies in total lysates, flow-through (FT) and the FLAG-immunoprecipitated fraction of Mia PaCa-2 cells expressing empty vector (EV) or FLAG-tagged RNF125 RM . ( D ) Interaction of endogenous RNF125 with HDAC2 in Mia PaCa-2 and KPC cells based on western blotting with an HDAC2 antibody in input and indicated immunoprecipitation fractions, and with or without the proteasome inhibitor MG132. ( E ) Western blot of RNF125 in input and indicated immunoprecipitated fractions showing interaction of endogenous RNF125 with HDAC2 in the cytoplasm (Cyt) and nucleus (Nuc) of Mia PaCa-2 cells. ( F) HDAC2 staining of normal pancreas from WT and Rnf125 −/− mice. Scale bar in low magnification panels, 250μm and in inset, 100 μm. ( G ) HDAC2 staining of control and shRNF125 KPC tumors. Scale bar in low magnification panels, 400μm and in inset, 50 μm. ( H ) HDAC2 staining in RNF125 Low vs. RNF125 High human PDA. Scale bar in low magnification panels, 600 μm and in inset, 150μm. ( I ) K63 HDAC2 ubiquitination of as analyzed in HEK293T cells overexpressing HDAC2, HA-Ub, and different RNF125 levels. IP, HDAC2; immunoblot, anti K63-linked Ub antibody.

Article Snippet: PDX1 (Addgene#114304, Addgene#114305), NR5A2 (DNASU#HsCD443475, Addgene#28093) and RFP-tagged histone H2B (Addgene#26001) expressing plasmids were obtained from Addgene or DNASU as indicated.

Techniques: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Transfection, Biomarker Discovery, Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Staining, Control, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Regulation of HDAC2-PDX1 by RNF125 defines pancreatic cancer development

doi: 10.1101/2020.01.06.896555

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of 7-day-old WT, single knockdown, or double knockdown KPC organoids probed with indicated antibodies. Scale bar, 100 μm. ( B ) Relative levels of indicated transcripts on PDX1 promoter (shown as % of input), in the ChIP performed in KPC cells. Shown are results represents 2 experiments. ( C ) ChIP analysis reveals relative enrichment of HDAC2 on the PDX1 promoter following RNF125 knockdown in KPC cells. Shown are representative data from 2 experiments. ( D ) Proliferation of indicated cell lines shown after transfection with indicated plasmids. One-way ANOVA was used to calculate statistical significance at the 96-hour time point. ( E ) Anchorage-dependent colony-forming assay of Mia PaCa2 cells after transfection with Scr control or two different shHDAC2 plasmids. ( F ) Western blot analysis of indicated proteins in Mia PaCa-2 cells expressing empty vector (EV) or RNF125 in the presence or absence of the K63R Ub mutant. ( G ) Pancreatic tumors 3 weeks after orthotopic injection of Scr-, shRNF125-, shHDAC2-, or shRNF125/shHDAC2-transduced KPC cells. n =8. Two-way ANOVA was used to calculate statistical significance. ( H ) RNF125, PDX1, or HDAC2 staining in tumors shown in (G). (I) Kaplan-Meier plots for HDAC2 High (upper) and HDAC2 Low (lower) PDA patients based on RNF125 levels. * P <0.05, ** P <0.01, *** P <0.005, **** P <0.001, NS – nonsignificant.

Article Snippet: PDX1 (Addgene#114304, Addgene#114305), NR5A2 (DNASU#HsCD443475, Addgene#28093) and RFP-tagged histone H2B (Addgene#26001) expressing plasmids were obtained from Addgene or DNASU as indicated.

Techniques: Immunofluorescence, Knockdown, Transfection, Control, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Injection, Staining

Journal: bioRxiv

Article Title: Regulation of HDAC2-PDX1 by RNF125 defines pancreatic cancer development

doi: 10.1101/2020.01.06.896555

Figure Lengend Snippet: ( A ) left, Immunoblotting with indicated antibodies and (right) qPCR analysis of PDX1 and NR5A2 transcripts 96 hours after transfection of Mia PaCa-2 or Panc-1 cells with Scr, shHDAC2#1, or shHDAC2#2 plasmids. qPCR results are means ± SEM in 2 independent experiments. Statistical significance was calculated using two-way ANOVA. ( B ) left, Ki67 stain in 48-hour-old KPC organoids transduced with Scr, shRNF125s, shHDAC2, or a shRNF125/shHDAC2 combination. right, Quantification of Ki67-positive nuclei. Statistical significance was calculated using one-way ANOVA. Scale bar, 100µm. ( C ) CK19, cleaved caspase-3, and Ki67 staining of orthotopic KPC tumors, either control or transduced with shRNF125, shHDAC2, or both. * P <0.05, ** P <0.01, *** P <0.005, **** P <0.001, NS – nonsignificant

Article Snippet: PDX1 (Addgene#114304, Addgene#114305), NR5A2 (DNASU#HsCD443475, Addgene#28093) and RFP-tagged histone H2B (Addgene#26001) expressing plasmids were obtained from Addgene or DNASU as indicated.

Techniques: Western Blot, Transfection, Staining, Transduction, Control