Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 1. Characteristics of the mutation c.303-305delGAA in TBX1. (A) Chromatograms of the homozygous mutation found in the TOF patient of cohort 1; (B) Structural representations of the mutation in TBX1 gene and protein; (C) Homology analysis of the 102 K position of TBX1 protein across different species; (D) the prediction result of MutationTaster. (*The probability value is the probability of the prediction, and a value close to 1 indicates a high ‘security’ of the prediction). (E) Chromatograms of the heterozygous c.303- 305delGAA mutation found in cohort 2.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Mutagenesis

Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 2. Subcellular localization of wild-type and mutant TBX1 in transiently transfected COS7 cells. Cells were co-stained with 4,6-diamino-2-phenylindole (DAPI) to illustrate nuclei. Wild-type TBX1 and the p.102delK mutant both localize to the nucleus.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Mutagenesis, Transfection, Staining

Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 3. The mutant protein TBX1102delK showed decreased expression level, and lost transcriptional activity. (A,B) Real-time qPCRs show the mRNA level of the mutant TBX1c.303_305delGAA was similar with that of the wild-type TBX1 in both 3T3 and C2C12 cells. (C,D) Western blots show the protein level of the mutant TBX1102delK and wild-type TBX1. The mutant showed reduced protein level compared with the wild-type in both 3T3 and C2C12 cells. The full-length blots are presented in the Supplementary Figure S1 and S2, respectively. (E,F) show that the 3T3 or C2C12 cells were co-transfected with the 4XT/2-minP reporter and either a pcDNA3.1(+) control vector (Blank), the TBX1 wild-type construct (WT), or the mutant TBX1c.303_305delGAA construct (Mut1). The results were normalized for transfection efficiency to a co-transfected pGL4.74[hRluc/ TK] vector. The TBX1102delK mutant showed significantly reduced transcription activity compared withthe wild- type protein in both 3T3 (reduced 42.4% vs. TBX1wt, p = 0.0112) and C2C12 cells (reduced 19.9% vs. TBX1wt, p = 0.0164). (G) Shows the C2C12 cells were co-transfected with the FGF10luc reporter and either the blank, the wild-type, or the mutant constructs (Mut1 = TBX1102delK and Mut2 = TBX1E129K). The results were normalized for transfection efficiency to a co-transfected pGL-TK vector. The TBX1102delK and TBX1E129K mutants all showed significantly reduced transcription activity compared with the wild-type protein (reduced 22.9% vs. TBX1wt, p = 0.0050 and reduced 28.7% vs. TBX1wt, p = 0.0112, respectively). (H) shows that significantly reduced transcriptional activity of the TBX1102delK and TBX1E129K mutant proteins on the WNTluc1 reporter compared with the wild-type protein in C2C12 cells (reduced 35.5% vs. TBX1wt, p = 0.0037 and reduced 25.4% vs. TBX1wt, p = 0.0156, respectively). (I) shows that the transcription activity of the two mutant proteins also decreased significantly on the WNTluc2 reporter in C2C12 cells (reduced 34% vs. TBX1wt, p = 0.0259 and reduced 31% vs. TBX1wt, p = 0.0242, respectively). The results are all shown as the mean ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; blank: pcDNA3.1(+) vector; WT: wild- type TBX1; mut1: the TBX1102delK mutant; mut2: the TBX1E129K mutant.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Mutagenesis, Expressing, Activity Assay, Western Blot, Transfection, Control, Plasmid Preparation, Construct

Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 4. Electrophoretic mobility shift assay to study DNA-binding activity of wild-type and mutant TBX1. DNA binding assay show that both the wild-type and p.102delK mutant TBX1 could bind to DNA (lane 2 and 3). Specificity of the binding is confirmed by nuclear protein of HEK293T cell transfected with the pcDNA3.1(+) control vector (lane 1) and the addition of unlabled probe (100X) (lane 4). The full-length image is presented in the Supplementary Figure S3.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay, Mutagenesis, DNA Binding Assay, Transfection, Control, Plasmid Preparation

Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 5. Proteasome inhibitor MG-132 increases both wild-type and mutant TBX1 protein level. C2C12 cells and 3T3 cells were transfected with equal concentrations of TBX1-encoding plasmids (the wild- type or the mutant 102delK) and treated with 20uM MG-132 or 10uM E-64 for 8 hours. The TBX1 levels were determined by Western blot (A: C2C12 cells; B: 3T3 cells). GAPDH was used as an internal control. n = 3. Wt: wild-type; Mut: the TBX1102delK mutant; vehicle: DMSO. The full-length image is presented in the Supplementary Figure S4.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Mutagenesis, Transfection, Western Blot, Control

Journal: EMBO Molecular Medicine

Article Title: FAK activity in cancer‐associated fibroblasts is a prognostic marker and a druggable key metastatic player in pancreatic cancer

doi: 10.15252/emmm.202012010

Figure Lengend Snippet: A Representative images of CK19 and α‐SMA immunohistochemistry and Sirius red staining in serial sections of primary mouse tumour (graft of FAK‐WT or FAK‐KD fibroblasts + tumours cells at 38 days after injection) at the invasive front. Scale bar, 100 μm. Bottom: schematic organization of the cells (tumour cells in brown, CAFs in yellow and non‐transformed epithelial cells in beige). B Representative images of ECM deposition (stained using Alexa Fluor 488 succinimidyl ester [NHS]) generated by FAK‐WT or FAK‐KD activated fibroblasts during migration. Scale bar, 50 μm. C Quantification of collagen I gel contraction (calculated based on photographed gels using the formula 100*(well diameter − gel diameter)/well diameter) induced by fibroblasts in presence or not of tumour cells (left: one‐way ANOVA with Tukey's method for multiple comparison), or by hCAFs in presence or not of FAK inhibitor (FAK‐I, 1 μM; right: paired two‐tailed Student's t ‐test for each time). Values are means ± SEM from three independent experiments of triplicates. * P < 0.05, ** P < 0.01, *** P < 0.001. D Quantification of the percentage change of Y397 FAK, total FAK, collagen I (coll I), collagen III (coll III), collagen IV (coll IV), LOXL2, periostin (POSTN), osteopontin (OPN) expression by human CAFs treated or not (NT) with FAK inhibitor (1 μM), based on immunofluorescence analyses. Values are means ± SEM obtained from at least five primary hCAFs, isolated from 5 to 12 different fresh human PDAC samples. NS: not significantly different; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 using paired two‐tailed Student's t ‐test. E Collagens I, III and IV and periostin deposition are shown using heat map of the IF staining, and cell shapes are identified by white line. Scale bar, 50 μm. F–K Characterization of the ECM‐enriched fraction (matrisome analyses) from hCAFs treated or not with FAK‐I by LC‐MS/MS proteomics. (F) Pie charts display the relative proportion of each of the major classes of matrisome proteins in non‐treated hCAFs ( N = 3 biological replicates with two technical replicates/condition) vs FAK-I‐treated hCAFs ( N = 3 biological replicates with two technical replicates/condition). (G, H) Left: Normalized peptide abundance for collagens (G) or proteoglycans (H) from hCAFs (each CAF duplicate is represented, CAF1 in blue, two in red and three in black) treated or not with FAK inhibitor. Right: Heatmap of the collagen (G) or proteoglycan (H) modifications in the three hCAFs upon FAK inhibition in hCAFs. The value of log 2 fold change between FAK inhibitor‐treated CAFs and non‐treated CAFs for each protein was indicated by the coloured scale, with red indicating increased expression while blue implies decreased expression. For each CAFs and each protein, log 2 fold change was calculated based on two technical replicates per condition (biological replicate n = 3, technical replicate n = 2). (I) Normalized peptide abundance for glycoproteins from hCAFs treated or not with FAK‐I. (J) Heatmap of the glycoprotein modifications upon FAK‐I in hCAFs. (K) Normalized peptide abundance for “matrisome‐associated” protein division: ECM‐affiliated proteins, secreted factors and ECM regulators from hCAFs treated or not with FAK inhibitor. (G, H, K) Each individual patient‐derived activated fibroblast was assigned to a specific symbol (see Table EV2 ). L Left: Representative pictures of β1 integrin activation (red) of tumour cells, upon adhesion to either ECM deposited from non‐treated CAFs or treated with FAK‐I for 7 days, analysed by IF. Merge: activated β1 integrin (red), phalloidin (green), dapi (blue). Right: quantification of the number of activated integrin β1 cluster per cells and size of those clusters. Values are means ± SEM, **** P < 0.0001 using paired two‐tailed Student's t ‐test on at least nine images (of three to 33 cells per image) per group, scale bar: 10 μm. Source data are available online for this figure.

Article Snippet: CAFs were isolated from human pancreatic tumour tissues using the outgrowth method described by Bachem et al ( ).

Techniques: Immunohistochemistry, Staining, Injection, Transformation Assay, Generated, Migration, Comparison, Two Tailed Test, Expressing, Immunofluorescence, Isolation, Liquid Chromatography with Mass Spectroscopy, Inhibition, Derivative Assay, Activation Assay

Journal: eLife

Article Title: Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts

doi: 10.7554/eLife.50663

Figure Lengend Snippet: ( A ) Unsupervised clustering of cytosine methylation profiles from seven primary patient-derived pancreatic cancer associated fibroblasts (CAFs) and four healthy controls (Hst: Hepatic stellate cells, Hdf: Human dermal fibroblasts, Hsf: Human skin fibroblasts) shows that CAFs are epigenetically distinct ( B ) Volcano plot shows that majority of differentially methylated loci in primary patient-derived CAFs are hypomethylated when compared to controls ( C ) Unsupervised clustering of gene expression profiles shows transcriptomic differences between CAFs and controls ( D ) Three independent MSCs were exposed to PANC-1 conditioned media (CM) for 21 days and analyzed for α-SMA ( ACTA1 ) and Fibroblast activation protein (FAP) expression. (T-test, p<0.05). ( E ) Unsupervised clustering based on cytosine methylation shows epigenomic differences between MSCs and de novo generated CAFs. Two independent experiments shown. ( F ) Volcano plot shows that the majority of differentially methylated loci in in vitro generated CAFs are hypomethylated ( G ) The differentially methylated regions in de novo generated CAFs are distributed throughout the genome as shown in the circos plot. H:Differentially methylated regions between MSC and de novo generated CAFs are present throughout the genome and mirror the distribution of HpaII sites in the genome. Hypo refers to Hypomethylated DMRs in CAFs. Hyper refers to Hypermethylated DMRs in CAFs when compared to MSCs. Genome refers to distribution of HpaII loci in the genome.

Article Snippet: Primary cultures of cancer associated fibroblasts (CAFs) were established from excess tissues of surgically resected pancreatic cancers at the Johns Hopkins Hospital.

Techniques: Methylation, Derivative Assay, Gene Expression, Activation Assay, Expressing, Generated, In Vitro

Journal: eLife

Article Title: Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts

doi: 10.7554/eLife.50663

Figure Lengend Snippet: ( A ) A set of 120 transcripts are commonly hypomethylated in primary patient-derived CAFs and de novo generated CAFs and includes CXCR4 . ( B,C ) The CXCR4 promoter is demethylated in primary patient-derived CAFs as seen by the HELP assay ( B ) and quantitative MassArray Epityper analysis ( C ). ( D - F ) CXCR4 knockdown in de novo CAFs leads to abrogation of the increased invasion of Panc1 cells on co-culture. (N = 3, p value<0.05) ( G ) Co-culture with de novo CAFs leads to increased transwell invasion by Panc-1 cells, that is abrogated after treatment of CAFs with CXCR4 inhibitor AMD-3100 (N = 3, p value<0.05) H: Gene expression profiling of CAFs with CXCR4 knockdown reveals signficantly downregulated ( left panel ) and upregulated ( right panel ) transcripts. Salient examples of downregulated transcripts include interleukins IL-8 and IL-33 and the chemokine CCL2.

Article Snippet: Primary cultures of cancer associated fibroblasts (CAFs) were established from excess tissues of surgically resected pancreatic cancers at the Johns Hopkins Hospital.

Techniques: Derivative Assay, Generated, HELP Assay, Knockdown, Co-Culture Assay, Gene Expression

Journal: eLife

Article Title: Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts

doi: 10.7554/eLife.50663

Figure Lengend Snippet: ( A ) CXCR4 siRNAs lead to knockdown of protein in CAFs when compared to control siRNAs. Western blot and image intensity is shown. ( B ) CXCR4 knockdown in dn-CAFs leads to abrogation of the increased invasion of Pa03C PDAC cells obseerved on co-culture. (N = 3, p value<0.05) ( C ) CXCR4 knockdown using a second set of siRNAs in dn-CAFs leads to abrogation of the increased invasion of Panc1 PDAC cells observed on co-culture. (N = 3, p value<0.05) ( D ) Co-culture with dn-CAFs leads to increased transwell invasion by Pa03C PDAC cells, which is abrogated after treatment of dn-CAFs with CXCR4 inhibitor AMD-3100 (N = 3, p value<0.05).

Article Snippet: Primary cultures of cancer associated fibroblasts (CAFs) were established from excess tissues of surgically resected pancreatic cancers at the Johns Hopkins Hospital.

Techniques: Knockdown, Control, Western Blot, Co-Culture Assay

Journal: eLife

Article Title: Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts

doi: 10.7554/eLife.50663

Figure Lengend Snippet: ( A ) Mapping of carbon atoms transition using U- 13 C 3 -lactate. Mass isotopomer distribution (MID) shows uptake of labelled lactate by MSCs cultured in α-MEM leading to the generation of α-KG (M2 αKG) via the Krebs cycle. ( B ) Conditioned media (CM) from mock treated and LDH inhibitor (FX11) treated Panc-1 (PDAC) cells was collected. MSCs were exposed to media alone, control conditioned media (PDAC CM), or LDH inhibitor treated conditioned media for 14 days in order to generate de novo CAFs (dn-CAF). TET enzymatic activity increases in dn-CAFs after exposure to PDAC CM and is abrogated after exposure to CM from LDH inhibitor treated Panc-1 cells (N = 2, p<0.05). ( C ) Quantitative analysis of 5-hMC levels by LC-MS demonstrates significant increase within dn-CAFs after treatment with Panc-1 CM, that is abrogated after exposure to CM from LDH inhibitor treated Panc-1 cells (N = 2, p<0.05). ( D ) CAFs were exposed to exogenous lactate and α-KG levels were analysed by ELISA. (N = 2, p<0.05) ( E ) 5hmC analysed in the resulting dn-CAFs by LC-MS. After a 2 week exposure to exogenous lactate, significantly increased 5hmC is observed in the dn-CAFs (N = 2, p<0.05). ( F ) Schematic model of lactate flux from tumor cells to MSCs during CAF differentiation, leading to aKG generation, TET activation and conversion of 5mC into 5hmC.

Article Snippet: Primary cultures of cancer associated fibroblasts (CAFs) were established from excess tissues of surgically resected pancreatic cancers at the Johns Hopkins Hospital.

Techniques: Cell Culture, Control, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: eLife

Article Title: Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts

doi: 10.7554/eLife.50663

Figure Lengend Snippet: ( A ) Conditioned media from ctrl and LDH siRNA mediated knockdown PANC-1 (PDAC) cells was collected. MSCs were exposed to control conditioned media (ctrl), or LDH knockdown conditioned and used to measure expression of CAF related genes (Fibroblasts specific protein, FSP1 ( S100A4 ); alpha smooth muscle actin (αSMA ( ACTA1 )); and vimentin (VIM) by qRTPCR. (N = 3, p<0.05). ( B ) TET activity was analysed in MSCs exposed to exogenous lactate in dose dependant fashion (1 mM to 10 mM). (N = 3, p<0.05). ( C ) TET activity was analysed in CAFs exposed to exogenous lactate (10 mM) with and without Pyruvate carrier inhibitor UK5099 (5 uM). (N = 3, p<0.05). ( D ) 5hmC and 5mC was analysed by ELISA in CAFs exposed to exogenous lactate with and without Pyruvate carrier inhibitor UK5099 (5 uM). (N = 2, p<0.05). E: TET activity was analysed in CAFs exposed to exogenous cell permeable αKG (2 mM). (N = 2, p<0.05).

Article Snippet: Primary cultures of cancer associated fibroblasts (CAFs) were established from excess tissues of surgically resected pancreatic cancers at the Johns Hopkins Hospital.

Techniques: Knockdown, Control, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts

doi: 10.7554/eLife.50663

Figure Lengend Snippet: ( A ) Genome wide 5hmC analysis was done by OXBS in MSCs and converted CAFs. 5hmC gains are depicted as percentage gained (CAF/MSC) and were seen to occur throughout the genome ( B ) 5hmC gains in CAFs were seen to occur at important genetic pathways C-G: 5hmC gains occurred with corresponding decrease in 5mC at genes related to CAF conversion (FSP1 ( S100A4 ), aSMA ( ACTA1 )); Collagen production (COL3A1), CXCR4, and the SMAD2.

Article Snippet: Primary cultures of cancer associated fibroblasts (CAFs) were established from excess tissues of surgically resected pancreatic cancers at the Johns Hopkins Hospital.

Techniques: Genome Wide

Journal: eLife

Article Title: Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts

doi: 10.7554/eLife.50663

Figure Lengend Snippet: ( A, B ) 5hmC ( A ) and CXCR4 ( B ) immunohistochemical staining was done on human PDAC TMAs and grading of intensity of stain in the tumor stromal CAFs was estimated. ( C ) Most CAF like cells in PDAC samples were positive for 5hmC and CXCR4 (1+ to 3+ intensity). Total PDAC samples examined for 5hmC were 254 and for CXCR4 were 261. ( D,E ) 5hmC ( D ) and CXCR4 ( E ) immunohistochemical staining was done on mouse PDAC tumors obtained from 3 KPC mice. CAF like stromal cells were positive for 5hmC and CXCR4. ( F ) Single cell RNA-seq from low grade intraductal papillary mucinous neoplasm (LG IPMN), high grade intraductal papillary mucinous neoplasm (HG IPMN) and frank pancreatic ductal adenocarcinoma (PDAC) was conducted and shown in tSne plot. ( G,H ) Stromal cell populations cluster distinctly and are positive for α-SMA ( ACTA1 ) and fibroblast activated protein. ( I ) Most of the stromal cells are seen in High grade IPMN and PDAC samples J,K: CXCR4 expression is seen in 14/181 (8%) stromal cells and correlates with cells with higher collagen expression, seen in activated CAF phenotypes (T-Test, P Vccal = 0.02).

Article Snippet: Primary cultures of cancer associated fibroblasts (CAFs) were established from excess tissues of surgically resected pancreatic cancers at the Johns Hopkins Hospital.

Techniques: Immunohistochemical staining, Staining, RNA Sequencing, Expressing