human bcl2  (Addgene inc)

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Diagram of original double-conditional allele ( Rosa BCL2+F ) showing FLAG-tagged human BCL2 driven by a constitutive promoter (CAG) and preceded by two stop transcriptional cassettes flanked by FRT and LoxP sites, respectively, followed by a stabilizing Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and inserted between left and right homology arms targeting the ROSA26 locus (R26L and R26R). Removal of the FRT-flanked stop cassette with Rosa Flp generates the Cre-dependent Rosa BCL2 allele. B. PCR genotyping of Rosa BCL2+F and Rosa BCL2 alleles validates removal of FRT-flanked stop cassette and insertion in the ROSA26 locus. C. Experimental design showing induction of BCL2 overexpression driven by Pdgfra CreER with tamoxifen (Tam) injection at P8 and evaluation during alveologenesis (P10-P21) and adulthood (P35 and 4 months). D. Immmunostaining and quantification of Pdgfra CreER/+ ; Rosa tdT/+ lungs showing efficient labeling of PDGFRA high AMFs (arrowhead), moderately MEOX2+ AF1s (open arrowhead), and occasionally HHIP+ DMFs (arrow: unlabeled DMFs). Lin: lineage. tdT level in AF1s is lower than that in AMFs and DMFs. AF2s in bronchovascular bundles are not included. Each symbol represents a mouse; average of three images of 424 x 424 um per mouse; error bar represents s.e.m. (see Table S1 for raw data; same for all figures unless noted otherwise). E. Immunostaining and quantification of Pdgfra CreER/+ ; Rosa BCL2/+ lungs showing comparable labeling efficiency and specificity with the Rosa tdT reporter. See D for details. F. Immunostaining of Pdgfra CreER/+ ; Rosa BCL2/tdT lungs, representative of at least 3 mice, showing FLAG and tdT colocalization. G. Immunostaining and quantification of Pdgfra CreER/+ ; Rosa BCL2/+ and control lungs showing that BCL2 overexpression reduces developmental AMF apoptosis (CASP3+PDGFRA high ). Asterisk: p=0.0087 (Mann-Whitney test). All scale bars: 20 um.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Virus, Over Expression, Injection, Labeling, Immunostaining, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Immunostaining of adult lungs shows that exogenous FLAG-tagged BCL2 activated by Shh Cre is specific to epithelial cells (NKX2-1+) and colocalizes with a mitochondrial marker TOMM22. Scale bar: 20 um. B. Webbing between digits four and five (arrowhead) from exogenous BCL2 activated by Shh Cre . C. Developmental defects including cleft palate (left), sacral spina bifida, and curly tail (right) from whole-embryo exogenous BCL2 activated by Sox2-Cre.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Immunostaining, Marker

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Immunostaining and quantification of mature Pdgfra CreER/+ ; Rosa tdT/+ control and Pdgfra CreER ; Rosa BCL2/tdT lungs showing more persistent MEOX2-tdT+ cells (arrow) in the BCL2 lung. The number of AF1s (MEOX2+) is unchanged (p=0.0635; Mann-Whitney test). Scale bar = 20 um (same for all panels unless noted otherwise). B. Immunostaining and quantification of mature Pdgfra CreER/+ ; Rosa BCL2/+ lungs showing three FLAG+ populations: expected AF1s (MEOX2+; open arrowhead) and aberrant DMF-like (HHIP+; arrowhead) and SCMF-like (MEOX2-HHIP-; arrow) cells. C. Immunostaining and quantification of mature Pdgfra CreER/+ ; Rosa BCL2/+ and control lungs showing more HHIP+ cells in BCL2 lungs (p=0.0043; Mann-Whitney test). Pie chart: in BLC2 lungs, 70.2% HHIP+ cells are DMF-like cells. D. Immunostaining and quantification of Pdgfra CreER/+ ; Rosa BCL2/+ lungs over time showing an increasing proportion of FLAG cells becoming DMF-like (HHIP+) and an increasing proportion of HHIP+ cells becoming lineage traced post developmental apoptosis of AMFs. E. Immunostaining and quantification of increased elastin fiber area (p=0.0317; Mann-Whitney test) in adult Pdgfra CreER/+ ; Rosa BCL2/+ and control lungs showing expansion of HHIP+ cells from proximal to distal alveolar ducts. DMF (FLAG-; asterisk) and DMF-like cells (FLAG+; arrow) are outlined by thick hydrazide-stained elastin fibers (arrowhead) while SCMF-like (FLAG+HHIP-; open arrow) are outlined by thin fibers (open arrowhead) upon BCL2 expression. Scale bar: 50 um

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Immunostaining, Control, MANN-WHITNEY, Staining, Expressing

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Immunostaining of mature lungs, representative of at least 4 mice (same below), shows that persistent cells (arrow) do not express PDGFRB or NOTCH3, markers of the vascular axis (open arrowhead). B. Immunostaining of neonatal and mature BCL2 lungs shows that neonatal AMFs (FLAG+PDGFRA high ; arrow in top row) are ACTA2+, while persistent AMFs (FLAG+; arrowhead in middle row) downregulate PDGFRA and ACTA2. Bottom row: DMF-like cells (FLAG+HHIP+; open arrowhead) and DMF (FLAG-HHIP+; open arrow) cells also downregulate TAGLN. C. Immunostaining of mature BCL2 lungs shows that DMF-like cells (FLAG+; arrowhead) co-express HHIP and CDH4, markers of DMFs (FLAG-; arrow). All scale bars: 20 um.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. UMAP and dot plot of mature lungs color-coded by cell types of epithelial, endothelial, mesenchymal, and immune lineages (top) and feature plots of Xist showing comparable results from male and female lungs (bottom). B. Volcano plots comparison of SCMF-like and DMF-like (Top), and DMF-like and DMFs (Bottom) from mature BCL2 lungs, showing upregulation of genes associated with stages of myofibroblasts maturation. Genes such as Pdgfra and ribosomal genes ( Rpl28 , Rpl23 , Rps19 ) found in SCMF-like and DMF-like represent immature and intermediate DMFs, while upregulation of DMF markers such as Fgf18 , Thbs2 , Myh11 , and Lum corresponds to more mature DMFs. C. Dot plots show that previously reported immature ( Zmat4 , Col24a1 , Tenm4 , Lef1 ) intermediate ( Mustn1 , Hhip , Thbs1 , Wnt5a ), and mature ( Scx , Lum , Aspn , Fhod3 ) myofibroblast markers of the developing lungs (P7 and P13) generally align with SCMF-like cells, DMF-like cells, and DMFs in BCL2 lungs. D. Volcano plot comparison of DMFs from P13 control and mature BCL2 lungs showing upregulation of mature DMF genes ( Lum and Dcn ) and genes associated with survival and differentiation ( Jak1 , Jak2 , Stat3 ) in BCL2 lungs. Upregulation of the lineage marker WPRE suggests that some persistent cells are reprogrammed completely into DMFs. E. Feature plots showing that like DMFs (arrow), DMF-like cells (arrowhead) mature by upregulating matrix genes Dcn and Lum and downregulating contractile genes Myh11 and Acta2.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Comparison, Control, Marker

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Top: UMAPs of lung mesenchymal cells color-coded by cell type (left) and axis (right) identify two clusters—DMF-like and SCMF-like—that are present only in Pdgfra CreER/+ ; Rosa BCL2/+ lungs (1 male and 1 female mice in each genotype). Bottom: Feature plots showing the lineage marker WPRE in AF1/2, DMF-like, and SCMF-like cells. Persistent AMFs unique to BCL2 lungs are negative for Meox2 and contain two subsets with opposing gradients of Pdgfra and Hhip, corresponding to SCMF-like and DMF-like cells in . Right: Dot plots of mesenchymal cell type markers showing that SCMF-like and DMF-like cells share AMF/DMF markers (Egfem1 and Tgfbi) but diverge for an AMF marker (Pdgfra) and DMF markers (Hhip, Cdh4, and Aspn). Fgf18 is higher in DMF-like cells than SCMF-like cells. B. Left: Monocle analysis of color-coded (as in A) mesenchymal cells of the epithelial axis from BCL2 lungs, showing a transcriptomic continuum from SCMF-like to DMF-like and then DMF cells. A bifurcation within DMFs, marked by increased contractile genes Actc1 and Acta2, corresponds to ASMCs or intermediate ASMC-DMF cells. Right: Trajectory heatmap showing ribosomal genes in SCMF-like cells, consistent with an immature state, and DMF markers in DMF-like cells. C. Top: Volcano plot comparison of mesenchymal cells of the epithelial axis from control and BCL2 lungs showing increased ribosomal genes in BCL2 lungs and decreased contractile genes and DMF markers, driven by SCMF-like cells. Bottom: Geno ontology analysis shows pathway enrichment in control (left) and BCL2 (right) lungs. See Table S2 for raw data (same for other scRNA-seq analyses). D. Integrated UMAPs of mesenchymal cells from normal lungs during alveologenesis and mature control and BCL2 lungs, showing that SCMF-like and DMF-like cells cluster with AMFs at P7 and P13. E. Volcano plot comparison of persistent AMFs (SCMF-like and DMF-like) from BCL2 lungs and AMFs from P13 lungs, showing enrichment of DMF and contractile markers, respectively. F. Feature (left) and violin (right) plots showing that the expression of the Seurat module score of ribosomal genes enriched in BCL2 lungs is higher in postnatal lungs compared to mature lungs (P20 and P38-control).

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Marker, Comparison, Control, Expressing

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Volcano plots comparison of epithelial, endothelial and immune lineage cells between P38 Pdgfra CreER/+ ; Rosa BCL2/+ and control lungs showing minimal changes in non-mesenchymal cell lineages. B. Left: CellChat ligand-receptor analysis of BCL2 lungs showing low outgoing and incoming interaction strength of SCMF-like and DMF-like cells. Right: Circle plots showing increasing interaction weights/strength for SCMF-like cells, DMF-like cells, and DMFs. C. Heatmap of interaction strength showing strongest interactions between DMFs and CAP1s, DMFs, pericytes, and AF2s. D. Differential incoming ligand-receptor interactions (Semaphorin and Bmp) of DMF-like cells, but not SCMF-like cells, with CAP1s, possibly mediating DMF-like reprogramming. E. Feature plots of neonatal and mature lungs show that expression of potential Semaphorin (Nrp1) and Bmp (Bmpr2) receptors mediating DMF-like reprogramming is higher in DMF-like cells (arrow) than SCMF-like cells (arrowhead). Plxna4, a receptor for Sema3c/3d, is specific for mesenchymal cells of the epithelial axis.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Comparison, Control, Expressing

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Immunostaining of acutely labeled lungs, representative of at least 4 mice, showing that Myh11-CreER targets AMFs (PDGFRA high ; arrow), but not AF1s (MEOX2+; arrowhead). B. Immunostaining of mature Myh11-CreER; Rosa BCL2/+ and control lungs, representative of at least 4 mice, showing more HHIP+ cells in BCL2 lungs extending to distal alveolar ducts. C. Immunostaining and quantification of mature Myh11-CreER; Rosa BCL2/+ and control lungs, showing the expansion of persistent populations FLAG+HHIP-PDGFRB-(SCMF-like; arrowhead) and FLAG+HHIP+ (DMF and DMF-like; arrow) in Myh11-CreER; Rosa Bcl2/+ lungs. This driver also targets pericytes (PDGFRB+FLAG+; open arrowhead). D. Top: UMAPs of lung mesenchymal cells from adult Myh11-CreER; Rosa BCL2/+ and Pdgfra CreE R/+ ; Rosa BCL2/+ and their respective control littermates showing comparable persistent cells (SCMF-like and DMF-like) with both drivers. Bottom: Feature plots show that the only cells targeted by both drivers (WPRE+) are DMF-like and SCMF-like cells, but not AF1/2 cells, pericytes, or A/VSMCs. E. Scatterplot comparison of average gene expression of AMFs (including SCMF-like and DMF-like cells; top) and DMFs (bottom) from Myh11-CreER; Rosa BCL2/+ and Pdgfra CreE R/+ ; Rosa BCL2/+ lungs, showing a high correlation (R =0.99) between drivers. All scale bars: 20 um.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Immunostaining, Labeling, Control, Comparison, Gene Expression

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Immunostaining and quantification of acutely labeled lungs showing that Pdgfrb CreER targets pericytes (PDGFRB+; arrowhead) and AMFs (PDGFRA High ; arrow), but not AF1s (MEOX2+; open arrowhead). B. Immunostaining of adult Pdgfrb CreER/+ ; Rosa BCL2/+ and control lungs shows more HHIP+ cells in BCL2 lungs in distal alveolar ducts. C. Top: UMAPs of lung mesenchymal cells from adult Pdgfrb CreE R/+ ; Rosa BCL2/+ and Pdgfra CreE R/+ ; Rosa BCL2/+ and their control littermates showing comparable persistent cells (SCMF-like and DMF-like) with both drivers. Bottom: Feature plots showing opposing gradients of Pdgfra and Hhip in targeted (WPRE+) SCMF-like and DMF-like cells, respectively, in both drivers. D. Scatterplot comparison of average gene expression of AMFs (including SCMF-like and DMF-like cells; top) and DMFs (bottom) from Pdgfrb CreE R/+ ; Rosa BCL2/+ and Pdgfra CreE R/+ ; Rosa BCL2/+ , showing a high correlation (R =0.97) between drivers. All scale bars: 20 um.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Immunostaining, Labeling, Control, Comparison, Gene Expression

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: A. Immunostained adult lungs exposed to 3 weeks of HDM showing upregulation of ACTA2 and TAGLN specifically in DMF (HHIP+; arrowhead), in comparison to PBS exposure. B. Immunostained adult control and Myh11-CreER; Rosa BCL2/tdT lungs exposed to 3 weeks of HDM showing that BCL2 lungs have an expansion of ACTA2+ cells to distal alveolar ducts, corresponding to DMF-like cells (HHIP+tdT+; comparing arrows in control and BCL2 lungs). C. Immunostained adult control and Myh11-CreER; Rosa BCL2/tdT lungs exposed to 3 weeks of HDM showing other lineage-labeled cells including SCMF-like cells (PDGFRB-tdT+; arrowhead) and pericytes (PDGFRB+tdT+; open arrowhead) do not express ACTA2. Arrow: ACTA2+tdT+ DMFs or DMF-like cells. D. Quantification showing comparable increase in ACTA2+ cells and the percentages of HHIP+ACTA2+ cells within HHIP+ or ACTA2+ alveolar cells in control and Myh11-CreER; Rosa BCL2/tdT lungs upon HDM exposure, in comparison to PBS exposure. Note that BCL2 lungs have more HHIP+ cells. All scale bars: 20 um.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Comparison, Control, Labeling

Journal: bioRxiv

Article Title: Apoptosis inhibition reprograms alveolar myofibroblasts toward ductal myofibroblasts

doi: 10.1101/2025.05.26.654588

Figure Lengend Snippet: Left: Schematics with color-coded cell types showing, from proximal to distal, ASMCs, DMFs and AMFs surrounding the lung epithelium. DMFs surround proximal alveolar ducts, while AMFs surround distal alveolar ducts and alveoli, and, upon BCL2 overexpression, become DMF-like (HHIP+) and SCMF-like (HHIP-) cells. Right: Neonatal AMFs and DMFs are contractile, and undergo apoptosis or become non-contractile, respectively. Upon HDM exposure, adult DMFs reactivate contractile genes, so do persistent DMF-like cells, but not persistent SCMF-like cells.

Article Snippet: The targeting vector Rosa BCL2+F was generated by replacing the tdT reporter in the Ai65 targeting vector (Addgene #61577) with FLAG-tagged human BCL2 (Addgene #8768).

Techniques: Over Expression

Journal: Particle and Fibre Toxicology

Article Title: Exposure to high dose of polystyrene nanoplastics causes trophoblast cell apoptosis and induces miscarriage

doi: 10.1186/s12989-024-00574-w

Figure Lengend Snippet: Supplement with Bcl-2 alleviated apoptosis in PS-NPs-exposed human trophoblast cells. ( A - C ) The mRNA and protein levels of Bcl-2 in Swan 71 cells with Bcl-2 overexpression. ( D - G ) The protein levels of Cleaved-caspase-2, Caspase-3, and Cleaved-caspase-3 in trophoblast cells exposed to 0 or 100 µg/mL PS-NPs and transfected with or without Bcl-2 overexpression plasmid for 24 h. ( H , I ) MMP levels in trophoblast cells exposed to 0 or 100 µg/mL PS-NPs and transfected with or without Bcl-2 overexpression plasmid for 1 h (scale bar, 20 μm). ( J , K ) Flow cytometry analysis and quantification of apoptosis rates (total early and late apoptosis) in trophoblast cells exposed to 0 or 100 µg/mL PS-NPs and transfected with or without Bcl-2 overexpression plasmid for 24 h

Article Snippet: Bcl-2 was also overexpressed by transfecting trophoblast cells with its overexpression plasmid (pcDNA3.1-Bcl-2), which was customized from Thermo Fisher Scientific Company.

Techniques: Over Expression, Transfection, Plasmid Preparation, Flow Cytometry