Journal: STAR Protocols

Article Title: Protocol for minicircle production for gene therapy without subsequent cleanup steps

doi: 10.1016/j.xpro.2025.103982

Figure Lengend Snippet: Gel electrophoresis of induction of mCherry-P2A- TBX4 minicircles with corresponding nanodrops Gel showing parental plasmid and two separate elutions of minicircle post-induction of mCherry-P2A-TBX4 showing clean productions. (A) shows gel electrophoresis of DNA ladder (Ladder), Parental Plasmid (PP) from preinduction miniprep and two separate elutions of minicircle (MC) from the GeneJet Maxiprep which correspond to minicircle 1 (MC1) and minicircle 2 (MC2). The minicircles are lower in size which correspond to successful induction and lack parental plasmid band noted demonstrating lack of contamination. (B) shows nanodrop quantifications from the two minicircle elutions. The minicircle elution corresponds the number on the nanodrop.

Article Snippet: TaqMan mouse Tbx4 probes , Thermo Scientific , Mm01299756_g1.

Techniques: Nucleic Acid Electrophoresis, Plasmid Preparation

Journal: STAR Protocols

Article Title: Protocol for minicircle production for gene therapy without subsequent cleanup steps

doi: 10.1016/j.xpro.2025.103982

Figure Lengend Snippet: qPCR results comparing mouse and human expression of TBX4 in NIH/3T3 cells NIH/3T3 cells were transfected with either Empty or mCherry-P2A-TBX4 minicircles and incubated for 72 h before RNA extraction and reverse transcription. Mouse and human TBX4 was analyzed in both Empty and mCherry-P2A-TBX4 minicircles compared to mouse beta-actin expression. Values above the line are p-values between compared conditions. Data are represented as mean ± SD. p -values were calculated using a two-tailed T-test. p < 0.05 was deemed significant.

Article Snippet: TaqMan mouse Tbx4 probes , Thermo Scientific , Mm01299756_g1.

Techniques: Expressing, Transfection, Incubation, RNA Extraction, Reverse Transcription, Two Tailed Test

Journal: STAR Protocols

Article Title: Protocol for minicircle production for gene therapy without subsequent cleanup steps

doi: 10.1016/j.xpro.2025.103982

Figure Lengend Snippet: Gel electrophoresis of induction of mCherry-P2A- TBX4 minicircles with corresponding nanodrops Gel showing parental plasmid and two separate elutions of minicircle post-induction of mCherry-P2A-TBX4 showing clean productions. (A) shows gel electrophoresis of DNA ladder (Ladder), Parental Plasmid (PP) from preinduction miniprep and two separate elutions of minicircle (MC) from the GeneJet Maxiprep which correspond to minicircle 1 (MC1) and minicircle 2 (MC2). The minicircles are lower in size which correspond to successful induction and lack parental plasmid band noted demonstrating lack of contamination. (B) shows nanodrop quantifications from the two minicircle elutions. The minicircle elution corresponds the number on the nanodrop.

Article Snippet: TaqMan human TBX4 probes , Thermo Scientific , Hs01057581_m1.

Techniques: Nucleic Acid Electrophoresis, Plasmid Preparation

Journal: STAR Protocols

Article Title: Protocol for minicircle production for gene therapy without subsequent cleanup steps

doi: 10.1016/j.xpro.2025.103982

Figure Lengend Snippet: qPCR results comparing mouse and human expression of TBX4 in NIH/3T3 cells NIH/3T3 cells were transfected with either Empty or mCherry-P2A-TBX4 minicircles and incubated for 72 h before RNA extraction and reverse transcription. Mouse and human TBX4 was analyzed in both Empty and mCherry-P2A-TBX4 minicircles compared to mouse beta-actin expression. Values above the line are p-values between compared conditions. Data are represented as mean ± SD. p -values were calculated using a two-tailed T-test. p < 0.05 was deemed significant.

Article Snippet: TaqMan human TBX4 probes , Thermo Scientific , Hs01057581_m1.

Techniques: Expressing, Transfection, Incubation, RNA Extraction, Reverse Transcription, Two Tailed Test

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: TBX4 is primarily expressed in myofibroblasts and pericytes. (A) TBX4 expression levels from a single cell RNA‐seq atlas. Dot color is indicative of expression level; size is percent of cells expressing TBX4 within that category; labels are different cell types, including five subcategories of fibroblast. (B) RT‐PCR results demonstrate that TBX4 is mainly expressed in mesenchymal origin cell lines—highest in early human lung fibroblasts (HLF) and pericytes (HLPC), lower in smooth muscle (PASMC), lowest in endothelial cells (PMVEC). (C) RNA‐Scope shows location of TBX4 RNA (red dots) and the pericyte marker Cox4i2 (white dots) with DAPI nuclear stain (blue) at postnatal Day 7. Red arrows are to draw attention to an epithelial ring (negative); yellow arrow indicates a blood vessel, likely primarily endothelium (also negative). At P7, much of the alveolar space is still mesenchyme, which we would expect to be positive. RT‐PCR, real‐time polymerase chain reaction.

Article Snippet: The TBX4 antibody for Western Blot was sc‐398903 (F‐12) (Santa Cruz Biotechnology, Dallas, TX USA).

Techniques: Expressing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, RNAscope, Marker, Staining, Real-time Polymerase Chain Reaction

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: RNA‐seq outcomes. (A) Principal components analysis. HLF and HLPC are well separated, and control and TBX4 knockdown are separated; the direction of separation is distinct in HLF and HLPC, indicating different effects in the two cell types. (B) Overrepresented ontology groups in 552 genes altered at p < 0.05 and more than 1.2x in both HLF and HLPC. (C) Significantly altered matrix genes ( p < 0.05) have both discordant and concordant change with TBX4 knockout between cell types. HLF and HLPC are each normalized to their own wild‐type control; HLF are indicated by blue points and arrows, HLPC by red. HLF, human lung fibroblasts; HLPC, human lung pericytes.

Article Snippet: The TBX4 antibody for Western Blot was sc‐398903 (F‐12) (Santa Cruz Biotechnology, Dallas, TX USA).

Techniques: RNA Sequencing, Control, Knockdown, Knock-Out

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: (A) The workflow strategy for combining results from RNA‐seq and TBX4 ChIP‐seq carried out in HLF and HLPC from control and TBX4KD. There are 555 genes with altered expression in both cell types, and which contain putative TBX4 binding sites by ChIP‐seq. Circle size is proportional to number of genes; colors are arbitrary. (B) Plot of false discovery rate and enrichment ratio for overrepresented gene ontology groups among the 555 genes found in (A) above. ChIP‐seq, chromatin immunoprecipitation sequencing; HLF, human lung fibroblasts; HLPC, human lung pericytes.

Article Snippet: The TBX4 antibody for Western Blot was sc‐398903 (F‐12) (Santa Cruz Biotechnology, Dallas, TX USA).

Techniques: RNA Sequencing, ChIP-sequencing, Control, Expressing, Binding Assay

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: Ontology groups significantly overrepresented in genes bound and regulated by TBX4 in human lung fibroblasts and human lung pericyte cells.

Article Snippet: The TBX4 antibody for Western Blot was sc‐398903 (F‐12) (Santa Cruz Biotechnology, Dallas, TX USA).

Techniques: Activity Assay, Transduction, Sublimation

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: (A) The workflow of ChIP‐seq and following data analysis (B, C) Pie charts show the distribution of TBX4 binding sites in fibroblasts (B) and in pericytes (C) according to peak location across different genomic regions in the human genome. ChIP‐seq, chromatin immunoprecipitation sequencing; HLF, human lung fibroblasts; HLPC, human lung pericytes.

Article Snippet: The TBX4 antibody for Western Blot was sc‐398903 (F‐12) (Santa Cruz Biotechnology, Dallas, TX USA).

Techniques: ChIP-sequencing, Binding Assay

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: Migration and BrdU proliferation assays by knocking down TBX4 in HLF. (A) Time‐lapse microscopy images of migration of knockdown (KD) control (left panels) and TBX4 KD (right panels) in HLF at 0, 24 h after scratch. The dotted lines define the area lacking cells. (B) Quantification of the migration distance during 24 h. (C) Quantitative analysis of BrdU‐positive HLF TBX4 KD control and TBX4 KD cells after incubated with BrdU 48 h. BrdU, bromodeoxyuridine; HLF, human lung fibroblasts.

Article Snippet: The TBX4 antibody for Western Blot was sc‐398903 (F‐12) (Santa Cruz Biotechnology, Dallas, TX USA).

Techniques: Migration, Time-lapse Microscopy, Knockdown, Control, Incubation

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: TBX4 is primarily expressed in myofibroblasts and pericytes. (A) TBX4 expression levels from a single cell RNA‐seq atlas. Dot color is indicative of expression level; size is percent of cells expressing TBX4 within that category; labels are different cell types, including five subcategories of fibroblast. (B) RT‐PCR results demonstrate that TBX4 is mainly expressed in mesenchymal origin cell lines—highest in early human lung fibroblasts (HLF) and pericytes (HLPC), lower in smooth muscle (PASMC), lowest in endothelial cells (PMVEC). (C) RNA‐Scope shows location of TBX4 RNA (red dots) and the pericyte marker Cox4i2 (white dots) with DAPI nuclear stain (blue) at postnatal Day 7. Red arrows are to draw attention to an epithelial ring (negative); yellow arrow indicates a blood vessel, likely primarily endothelium (also negative). At P7, much of the alveolar space is still mesenchyme, which we would expect to be positive. RT‐PCR, real‐time polymerase chain reaction.

Article Snippet: For TBX4 knockdown (KD) in HLF by TBX4 siRNA, cells were plated at a density of 1.8 × 10 6 cells/plate in a 10 cm plate, and then transfected with 150 pmol TBX4 siRNA or control siRNA by Lipofectamine RNAiMAX reagent (Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.

Techniques: Expressing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, RNAscope, Marker, Staining, Real-time Polymerase Chain Reaction

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: RNA‐seq outcomes. (A) Principal components analysis. HLF and HLPC are well separated, and control and TBX4 knockdown are separated; the direction of separation is distinct in HLF and HLPC, indicating different effects in the two cell types. (B) Overrepresented ontology groups in 552 genes altered at p < 0.05 and more than 1.2x in both HLF and HLPC. (C) Significantly altered matrix genes ( p < 0.05) have both discordant and concordant change with TBX4 knockout between cell types. HLF and HLPC are each normalized to their own wild‐type control; HLF are indicated by blue points and arrows, HLPC by red. HLF, human lung fibroblasts; HLPC, human lung pericytes.

Article Snippet: For TBX4 knockdown (KD) in HLF by TBX4 siRNA, cells were plated at a density of 1.8 × 10 6 cells/plate in a 10 cm plate, and then transfected with 150 pmol TBX4 siRNA or control siRNA by Lipofectamine RNAiMAX reagent (Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.

Techniques: RNA Sequencing, Control, Knockdown, Knock-Out

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: (A) The workflow strategy for combining results from RNA‐seq and TBX4 ChIP‐seq carried out in HLF and HLPC from control and TBX4KD. There are 555 genes with altered expression in both cell types, and which contain putative TBX4 binding sites by ChIP‐seq. Circle size is proportional to number of genes; colors are arbitrary. (B) Plot of false discovery rate and enrichment ratio for overrepresented gene ontology groups among the 555 genes found in (A) above. ChIP‐seq, chromatin immunoprecipitation sequencing; HLF, human lung fibroblasts; HLPC, human lung pericytes.

Article Snippet: For TBX4 knockdown (KD) in HLF by TBX4 siRNA, cells were plated at a density of 1.8 × 10 6 cells/plate in a 10 cm plate, and then transfected with 150 pmol TBX4 siRNA or control siRNA by Lipofectamine RNAiMAX reagent (Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.

Techniques: RNA Sequencing, ChIP-sequencing, Control, Expressing, Binding Assay

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: Ontology groups significantly overrepresented in genes bound and regulated by TBX4 in human lung fibroblasts and human lung pericyte cells.

Article Snippet: For TBX4 knockdown (KD) in HLF by TBX4 siRNA, cells were plated at a density of 1.8 × 10 6 cells/plate in a 10 cm plate, and then transfected with 150 pmol TBX4 siRNA or control siRNA by Lipofectamine RNAiMAX reagent (Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.

Techniques: Activity Assay, Transduction, Sublimation

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: (A) The workflow of ChIP‐seq and following data analysis (B, C) Pie charts show the distribution of TBX4 binding sites in fibroblasts (B) and in pericytes (C) according to peak location across different genomic regions in the human genome. ChIP‐seq, chromatin immunoprecipitation sequencing; HLF, human lung fibroblasts; HLPC, human lung pericytes.

Article Snippet: For TBX4 knockdown (KD) in HLF by TBX4 siRNA, cells were plated at a density of 1.8 × 10 6 cells/plate in a 10 cm plate, and then transfected with 150 pmol TBX4 siRNA or control siRNA by Lipofectamine RNAiMAX reagent (Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.

Techniques: ChIP-sequencing, Binding Assay

Journal: Pulmonary Circulation

Article Title: RNA‐Seq and ChIP‐Seq Identification of Unique and Overlapping Target Genes and Pathways Regulated by TBX4 in Human Pulmonary Fibroblasts and Pericytes

doi: 10.1002/pul2.70058

Figure Lengend Snippet: Migration and BrdU proliferation assays by knocking down TBX4 in HLF. (A) Time‐lapse microscopy images of migration of knockdown (KD) control (left panels) and TBX4 KD (right panels) in HLF at 0, 24 h after scratch. The dotted lines define the area lacking cells. (B) Quantification of the migration distance during 24 h. (C) Quantitative analysis of BrdU‐positive HLF TBX4 KD control and TBX4 KD cells after incubated with BrdU 48 h. BrdU, bromodeoxyuridine; HLF, human lung fibroblasts.

Article Snippet: For TBX4 knockdown (KD) in HLF by TBX4 siRNA, cells were plated at a density of 1.8 × 10 6 cells/plate in a 10 cm plate, and then transfected with 150 pmol TBX4 siRNA or control siRNA by Lipofectamine RNAiMAX reagent (Invitrogen Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.

Techniques: Migration, Time-lapse Microscopy, Knockdown, Control, Incubation