Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A) Schematic of directed differentiation protocol to generate hiEndos. (B) Phase contrast images during the indicated day of differentiation. White dashed square represents region magnified in insets. Scale bar: 50um. (C-E) RT-qPCR for the indicated primers using RNA isolated from cells on each day of differentiation, purified hiEndos at P0 and P5, and HUVECs. N=4 experimental replicates from independent wells of the same differentiation, representative results from two independent differentiations. (F) Flow cytometry of hiEndos using antibodies against the human endothelial lineage markers CD31 and CD144 (right) and isotype controls (left), N=4 biological replicates from distinct differentiations. (G) Immunostaining of hiEndos for human CD31 (green, left) and CD144 (red, right). Hoechst stains nuclei (blue). Scale bar 10um. N=3 biological replicates. (H) Capillary tube formation assay on 3D Matrigel. Scale bar 50um. N=4 biological replicates. (I-J) Human CD31 and acetylated LDL uptake in hiEndos measured by flow cytomtetry (I) or fluorescence microscopy (J). Scale bar 50um. N=8 biological replicates. (K) Schematic of experiment to test the impact of Notch (DAPT) and TGF-beta (SB431542) inhibition on hiEndo expansion (top). Quantification of CD31 and CD144 double-positive cells by flow cytometry (left) and cumulative hiEndo yield (right) at each passage grown in the indicated media conditions. N=2 experimental replicates from independent wells of the same differentiation, representative data shown from 3 independent differentiations. (L) Quantification of total hiEndo yield per input hiEndo in each media. ** p<0.01 using one-way Anova and Tukey’s multiple comparison test. (M) RT-qPCR of arterial and venous markers after serial passaging in SD medium. N=4 experimental replicates from independent wells of the same differentiation, representative results from two independent differentiations. ** p<0.01, *** p<0.001, **** p<0.0001, one-way Anova and Tukey’s multiple comparison test. Abbreviations: hiPSC (human induced pluripotent stem cell), CHIR (CHIR99021), P (passage), HUVEC (human umbilical vein endothelial cells), AcLDL (acetylated low-density lipoprotein), SB (SB431542).

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Quantitative RT-PCR, Isolation, Purification, Flow Cytometry, Immunostaining, Capillary Tube Formation Assay, Fluorescence, Microscopy, Inhibition, Comparison, Passaging

Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A-C) Fold change in the expression of indicated transcripts in hiPSC-derived cells (BU3 clone) on each day of hiEndo differentiation, in purified hiEndos at P0 and P5, and in HUVECs (RT-qPCR). N=4 experimental replicates from independent wells of the same differentiation. (D) Quantitation of hiEndo differentiation efficiency on day 5 of differentiation across 12 distinct differentiations. (E) Flow cytometry of human CD31, human CD144, and isotype controls pre- and post-MACs purification of hiEndos on day 5 of the differentiation. N=1. (F) Phase contrast microscopy of BU3 hiEndos showing cobblestone morphology. Scale bar 50um. (G) Flow cytometry of human CD31, human CD44, and isotype controls in BU3 hiEndos, the plot is representative of 4 experimental replicates of independent wells from the same differentiation. (H) Quantification of the percent of CD31/CD144 double positive hiEndos by flow cytometry at each passage. N=1 differentiation (BU1). (I) Phase contrast microscopy of BU1 hiEndos at sequential passages. Scale bar 50um. Results are representative of 2 experimental replicates of independent wells from the same differentiation. (J) RT-qPCR analysis of fold change in transcript expression of markers of fibroblast and smooth muscle identity in hiEndos at sequential passages. N=2 experimental replicates of independent wells from the same differentiation. **** p<0.0001 by one-way Anova with Tukey’s multiple comparisons.

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Expressing, Derivative Assay, Purification, Quantitative RT-PCR, Quantitation Assay, Flow Cytometry, Microscopy

Journal: Scientific Reports

Article Title: YBX1/PPIB axis promotes post-maturation arteriovenous fistula stenosis via enhancing endothelial to mesenchymal transition

doi: 10.1038/s41598-026-49568-y

Figure Lengend Snippet: Identification of AVFS-Associated Cell Subpopulations and Candidate Genes. (A) UMAP plot showing the grouping of 10 cell types. (B) Expression of key marker genes across cell types. (C) Differential proportions of each cell type between AVFS and non-stenotic control. (D) Volcano plot of DEGs in endothelial cells. (E-G) GO, KEGG and Reactome enrichment analysis illustrating predominant biological functions and pathways. (H) Flow cytometry sorting and analysis of endothelial cells from vascular tissues, n = 3. (I) qRT-PCR analysis of PPIB, CD31, VE-cadherin, and α-SMA expression levels in endothelial cells from AVFS and non-stenosis tissues, n = 3. (J) WB analysis of PPIB, CD31, VE-cadherin, and α-SMA expression levels in endothelial cells from AVFS and non-stenosis tissues, n = 3. Data are presented as mean ± SD, differences between groups were analyzed by paired t-test. * P < 0.05; **<0.01; ***<0.001; ****<0.0001. SMC: Smooth muscle cell, EC: Endothelial cell, GO: Gene Ontology, KEGG: Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: The antibodies used included: anti-PPIB antibody (CUSABIO, China; #CSB-PA018472ESR1HU), anti-YBX1 antibody (Abclonal, China; #A3534), anti-VE-cadherin antibody (Abclonal, China; #A12416), anti-CD31 antibody (Abclonal, China; #A19014), anti-α-SMA antibody (Affinity, China, #AF1032), and anti-GAPDH antibody (Abclonal, China; #A19056).

Techniques: Expressing, Marker, Control, Flow Cytometry, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: YBX1/PPIB axis promotes post-maturation arteriovenous fistula stenosis via enhancing endothelial to mesenchymal transition

doi: 10.1038/s41598-026-49568-y

Figure Lengend Snippet: PPIB Regulates Endothelial Marker in HUVECs. (A) qRT-PCR of PPIB, VE-cadherin, and CD31 expression following PPIB knockdown in HUVECs ( n = 3). (B) qRT-PCR analysis of PPIB, VE-cadherin, and CD31 expression following PPIB overexpression in HUVECs ( n = 3). (C) Western blot of PPIB, VE-cadherin and CD31 after PPIB knockdown in HUVECs ( n = 3). (D) Western blot of PPIB, VE-cadherin, and CD31 expression after PPIB overexpression in HUVECs ( n = 3). The protein bands of WB were derived from different gels, and the uncropped original WB images are provided in the Supplementary Material 1. Data are presented as mean ± SD, differences between groups were analyzed by one-way ANOVA, statistical significance was determined using one-way ANOVA. Compared to the control group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; compared to the TGF-β1 group, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001. sh: short hairpin, gene expression interference by introducing short hairpin RNA. oe: overexpression, increasing gene expression by introducing the target gene sequence. NC: Negative control, for sh, refers to lentivirus construction by transfecting plasmids with a scrambled shRNA sequence; for oe, refers to lentivirus construction by directly transfecting empty plasmids. ns: not significant.

Article Snippet: The antibodies used included: anti-PPIB antibody (CUSABIO, China; #CSB-PA018472ESR1HU), anti-YBX1 antibody (Abclonal, China; #A3534), anti-VE-cadherin antibody (Abclonal, China; #A12416), anti-CD31 antibody (Abclonal, China; #A19014), anti-α-SMA antibody (Affinity, China, #AF1032), and anti-GAPDH antibody (Abclonal, China; #A19056).

Techniques: Marker, Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Western Blot, Derivative Assay, Control, Gene Expression, shRNA, Sequencing, Negative Control

Journal: Scientific Reports

Article Title: YBX1/PPIB axis promotes post-maturation arteriovenous fistula stenosis via enhancing endothelial to mesenchymal transition

doi: 10.1038/s41598-026-49568-y

Figure Lengend Snippet: Transcription Factor YBX1 is Associated with EndMT in Endothelial Cells. (A) Prediction of YBX1 binding sites in the PPIB promoter and schematic of site-directed mutations used in the dual-luciferase assay. (B) Dual-luciferase assay validating the interaction between YBX1 and the PPIB promoter region ( n = 3). (C) qRT-PCR analysis of YBX1, PPIB, VE-cadherin, and CD31 expression levels ( n = 3). (D–E) Western blot of YBX1, PPIB, VE-cadherin, and CD31 expression levels ( n = 3). (F , H) Immunofluorescence staining assessing α-SMA expression levels ( n = 3). (G , I) Wound healing assays evaluating HUVEC migration ability ( n = 3). The protein bands of WB were derived from different gels, and the uncropped original WB images are provided in the Supplementary Material 1. Data are presented as mean ± SD, differences between groups were analyzed by one-way ANOVA, statistical significance was determined using one-way ANOVA. * P < 0.05, **< 0.01, ***< 0.001, ****< 0.0001. luc: Firefly luciferase. RL: Renilla luciferase. WT: Wild type. Mut: Mutant. oe: overexpression, increasing gene expression by introducing the target gene sequence. NC: Negative control, for oe, refers to lentivirus construction by directly transfecting empty plasmids. ns: not significant.

Article Snippet: The antibodies used included: anti-PPIB antibody (CUSABIO, China; #CSB-PA018472ESR1HU), anti-YBX1 antibody (Abclonal, China; #A3534), anti-VE-cadherin antibody (Abclonal, China; #A12416), anti-CD31 antibody (Abclonal, China; #A19014), anti-α-SMA antibody (Affinity, China, #AF1032), and anti-GAPDH antibody (Abclonal, China; #A19056).

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Migration, Derivative Assay, Mutagenesis, Over Expression, Gene Expression, Sequencing, Negative Control

Journal: Scientific Reports

Article Title: YBX1/PPIB axis promotes post-maturation arteriovenous fistula stenosis via enhancing endothelial to mesenchymal transition

doi: 10.1038/s41598-026-49568-y

Figure Lengend Snippet: YBX1 Promotes EndMT through Regulation of PPIB. (A) qRT-PCR assessed the expression levels of YBX1, PPIB, VE-cadherin, and CD31 in each group, n = 3. (B) Western blot assessed the expression levels of YBX1, PPIB, VE-cadherin, and CD31 in each group, n = 3. (C) Immunofluorescence staining of α-SMA expression in HUVECs from each group ( n = 3). (D) Wound healing assay evaluating HUVEC migratory capacity in each group ( n = 3). The protein bands of WB were derived from different gels, and the uncropped original WB images are provided in the Supplementary Material 1. Data are presented as mean ± SD, differences between groups were analyzed by one-way ANOVA, statistical significance was determined using one-way ANOVA. * P < 0.05, **<0.01, ***<0.001, ****<0.0001 (vs. TGF-β1 group); # P < 0.05, ##<0.01, ###<0.001, ####<0.0001 (vs. YBX1-oe + NC-sh + TGF-β1 group); ^ P < 0.05, ^^<0.01, ^^^<0.001, ^^^^<0.0001 (vs. NC-oe+PPIB-sh + TGF-β1 group). sh: short hairpin, gene expression interference by introducing short hairpin RNA. oe: overexpression, increasing gene expression by introducing the target gene sequence. NC: Negative control, for sh, refers to lentivirus construction by transfecting plasmids with a scrambled shRNA sequence; for oe, refers to lentivirus construction by directly transfecting empty plasmids. ns: not significant.

Article Snippet: The antibodies used included: anti-PPIB antibody (CUSABIO, China; #CSB-PA018472ESR1HU), anti-YBX1 antibody (Abclonal, China; #A3534), anti-VE-cadherin antibody (Abclonal, China; #A12416), anti-CD31 antibody (Abclonal, China; #A19014), anti-α-SMA antibody (Affinity, China, #AF1032), and anti-GAPDH antibody (Abclonal, China; #A19056).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Wound Healing Assay, Derivative Assay, Gene Expression, shRNA, Over Expression, Sequencing, Negative Control