Journal: Oncogenesis

Article Title: P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker.

doi: 10.1038/s41389-024-00541-2

Figure Lengend Snippet: Fig. 3 P4HB maintains GSCs stemness via the Wnt/β-Catenin signaling pathway. A Relative mRNA expression levels of the indicated signaling pathways in 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentiviral vectors. Data are presented as means ± SD, n = 3, t-test. B–D Western blot analyses of P4HB, β-catenin, and Cyclin D1 protein levels in X01, 448, 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus, with α-tubulin used as a loading control. E Western blot analysis of P4HB and β-catenin in isolated nuclear or cytosolic lysates from 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus. Lamin B1 and β-actin were used as markers for the nucleus and cytoplasm, respectively. F Heatmap analysis of different gene expression clusters in X01 shCtrl and shP4HB GSCs by RNAseq analysis. G Differentially expressed genes were mapped by fold change between the shCtrl and shP4HB groups. H Western blot analysis of P4HB and LRP6 protein levels in X01 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus, Vinculin was used as a loading control. I Western blot analysis of β-catenin, CyclinD1, and LRP6, P-LRP6 protein levels 3 in X01 and 83 GSCs treated with 10 μM ICG001, 25 ng/ml recombinant P4HB protein (rP4HB). GAPDH was used as a loading control.

Article Snippet: The recombinant protein was purchased from MCE (HY-P71917).

Techniques: Expressing, Protein-Protein interactions, Infection, Western Blot, Control, Isolation, Gene Expression, RNA sequencing, Recombinant

Journal: Journal of Nuclear Medicine

Article Title: Molecular Imaging of Fibroblast Activity After Myocardial Infarction Using a 68 Ga-Labeled Fibroblast Activation Protein Inhibitor, FAPI-04

doi: 10.2967/jnumed.119.226993

Figure Lengend Snippet: (A) Tile scan of entire infarcted heart section showing location of FAP+ fibroblasts. Insets 1, 2, and 3 show higher magnification from infarct border, infarct center, and noninfarcted remote myocardium, respectively. (B) Enumeration of FAP+ fibroblast density in MI hearts (n = 3) showing higher FAP+ fibroblast percentage in border zone than in infarct center or remote zone. (C) H&E-stained parallel section. (D) Photomicrographs of FAP, prolyl-4-hydroxylase β (P4H), α-smooth muscle actin, and vimentin-stained infiltrated fibroblasts in periinfarct border zone. Abundant colocalization of FAP, prolyl-4-hydroxylase β, and vimentin shows that accumulated fibroblasts are activated phenotypes, whereas small portion have differentiated into α-smooth muscle actin–positive mature myofibroblasts.

Article Snippet: Tissue sections were stained with antibodies against FAP (R&D Systems) as a marker of activated fibroblasts, prolyl-4-hydroxylase β (Origene) as a marker of activated and collagen-synthesizing fibroblasts, α-smooth muscle actin (Merck) as a marker of differentiated mature myofibroblasts, and vimentin (Merck) as a general fibroblast marker, as described previously ( 8 ).

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Microbiota dynamics, metabolic and immune interactions in the cervicovaginal environment and their role in spontaneous preterm birth

doi: 10.3389/fimmu.2023.1306473

Figure Lengend Snippet: A summary of bacteriocins produced by bacteria from the female genital tract, their molecular weight, and mode of action.

Article Snippet: Non-pregnant 50 women with BV (18-55 yrs) , Postbiotic gel Lacticaseibacillus paracasei ProSci-92 Lacticaseibacillus rhamnosus ProSci-109 1 × 10 6 cfu/mL Daily for 7 days 1 capsule/o.d./7d , China , -relative abundance of vaginal lactobacilli increased compared to baseline -reduced Gardnerella , Prevotella , and Atopobium , ( ) .

Techniques: Produced, Bacteria, Molecular Weight, Membrane

Journal: Oncogenesis

Article Title: P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker

doi: 10.1038/s41389-024-00541-2

Figure Lengend Snippet: A – C Kaplan–Meier survival curves for patients with glioma from the TCGA, CGGA, and REMBRANDT datasets, stratified by high and low expression of P4HB . P -values were calculated using the log-rank test and Wilcoxon test. D UMAP plot showing the clustering of different cell types within the GBM patients’ tissues, and the expression levels of P4HB across different cell clusters. E – G Relative mRNA expression levels of P4HB in X01, 448, and 83 GSCs, respectively, were measured by qRT-PCR after shRNA-mediated knockdown of P4HB . Data are presented as means ± SD, n = 3, *** P < 0.001, t-test. H Western blot analysis of P4HB, NESTIN, and SOX2 protein levels in X01 GSCs after shRNA-mediated knockdown of P4HB , α-tubulin was used as a loading control. I Limit-dilution assay for sphere-forming capacity in X01 GSCs following shRNA-mediated knockdown of P4HB . Log fraction without spheres is plotted against the number of initial cells per well. ** P < 0.01, *** P < 0.001, t-test. J Western blot analysis of P4HB, NESTIN, and SOX2 protein levels in 448 GSCs after shRNA-mediated knockdown of P4HB , α-tubulin was used as a loading control. K Limiting dilution assay for sphere-forming capacity in 448 GSCs following shRNA-mediated knockdown of P4HB . ** P < 0.01, *** P < 0.001, t-test. L Western blot analysis of P4HB, NESTIN, and CD44 protein levels in 83 GSCs after shRNA-mediated knockdown of P4HB , α-tubulin was used as a loading control. M Limiting dilution assay for sphere-forming capacity in 83 GSCs following shRNA-mediated knockdown of P4HB . ** P < 0.01, *** P < 0.001, t-test.

Article Snippet: V-ZAD-FMK, Z-DEVE-FMK dimethyl fumarate, ferrostatin-1, and the recombinant P4HB protein were purchased from MedChemExpress.

Techniques: Expressing, Quantitative RT-PCR, shRNA, Knockdown, Western Blot, Control, Dilution Assay, Limiting Dilution Assay

Journal: Oncogenesis

Article Title: P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker

doi: 10.1038/s41389-024-00541-2

Figure Lengend Snippet: A – C Cell proliferation assay for X01, 448, and 83 GSCs infected with shCtrl, shP4HB-1, and shP4HB-2 lentiviral vectors. Data are presented as means ± SD, n = 3, * P < 0.05, *** P < 0.001, t-test. D Kaplan–Meier survival curves of mice implanted with 83 GSCs infected with shCtrl or shP4HB lentivirus ( n = 6 in each group, 1 × 10 4 GSCs/mouse), P < 0.0001, log-rank test. E H&E staining of the whole brain slides derived from the mice implanted with 83 GSCs infected with shCtrl or shP4HB lentivirus. F – H Flow cytometry analysis of Annexin V/PI staining for cellular apoptosis analysis of X01, 448, 83 GSCs infected with shCtrl or shP4HB lentivirus. I – K Western blot analysis of PARP, C - PARP, P4HB, Caspase-3, CC3, and BCL2 protein levels in X01, 448, 83 GSCs infected with shCtrl or shP4HB lentivirus, α-tubulin was used as a loading control.

Article Snippet: V-ZAD-FMK, Z-DEVE-FMK dimethyl fumarate, ferrostatin-1, and the recombinant P4HB protein were purchased from MedChemExpress.

Techniques: Proliferation Assay, Infection, Staining, Derivative Assay, Flow Cytometry, Western Blot, Control

Journal: Oncogenesis

Article Title: P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker

doi: 10.1038/s41389-024-00541-2

Figure Lengend Snippet: A Relative mRNA expression levels of the indicated signaling pathways in 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentiviral vectors. Data are presented as means ± SD, n = 3, t-test. B – D Western blot analyses of P4HB, β-catenin, and Cyclin D1 protein levels in X01, 448, 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus, with α-tubulin used as a loading control. E Western blot analysis of P4HB and β-catenin in isolated nuclear or cytosolic lysates from 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus. Lamin B1 and β-actin were used as markers for the nucleus and cytoplasm, respectively. F Heatmap analysis of different gene expression clusters in X01 shCtrl and shP4HB GSCs by RNAseq analysis. G Differentially expressed genes were mapped by fold change between the shCtrl and shP4HB groups. H Western blot analysis of P4HB and LRP6 protein levels in X01 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus, Vinculin was used as a loading control. I Western blot analysis of β-catenin, CyclinD1, and LRP6, P-LRP6 protein levels 3 in X01 and 83 GSCs treated with 10 μM ICG001, 25 ng/ml recombinant P4HB protein (rP4HB). GAPDH was used as a loading control.

Article Snippet: V-ZAD-FMK, Z-DEVE-FMK dimethyl fumarate, ferrostatin-1, and the recombinant P4HB protein were purchased from MedChemExpress.

Techniques: Expressing, Protein-Protein interactions, Infection, Western Blot, Control, Isolation, Gene Expression, Recombinant

Journal: Oncogenesis

Article Title: P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker

doi: 10.1038/s41389-024-00541-2

Figure Lengend Snippet: A Schematic illustration of CCF642 inhibiting P4HB reductase enzyme activity by sheltering the active enzyme site. B Cell proliferation assays for X01 GSCs treated with different doses of CCF642 after 24-h treatments. Data are presented as means ± SD, n = 3, *** P < 0.001, t-test. C Western blot analysis of stemness markers NESTIN and SOX2 protein level in X01 GSCs after treatment with 1 μM CCF642 for 48 h, Vinculin was used as a loading control. D Limit-dilution assay for sphere-forming capacity in X01 GSCs treated with 0.1 μM CCF642. *** P < 0.001, t-test. E Flow cytometry analysis of cellular apoptosis of X01 GSCs treated with different doses of CCF642 by Annexin V/PI staining. F Western blot analysis of PARP, C-PARP, P4HB, Caspase-3, CC3, and BCL2 protein levels in X01 GSCs treated with 1 μM CCF642 under different time points, α-tubulin was used as a loading control. G Western blot analysis of PARP, C-PARP, P4HB, Caspase-3, and CC3 protein levels in X01 GSCs treated with 1 μM CCF642 in the presence of 25 μM V-ZAD-FMK, α-tubulin was used as a loading control. H Cell proliferation assays for X01 GSCs treated with 1 μM CCF642 along with apoptosis inhibitor V-ZAD-FMK (25 μM). Data are presented as means ± SD, n = 3, *** P < 0.001, t-test. I – K Western blot analysis of P4HB, β-catenin, CyclinD1, and LRP6 protein levels in X01, 448, 83 GSCs treated with 1 μM CCF642, α-tubulin was used as a loading control. L Western blot analysis of β-catenin protein level in fractionated nuclear or cytosolic lysates from 83 GSCs treated with 1 μM CCF642, α-tubulin, and LaminB1 was used as loading control, respectively.

Article Snippet: V-ZAD-FMK, Z-DEVE-FMK dimethyl fumarate, ferrostatin-1, and the recombinant P4HB protein were purchased from MedChemExpress.

Techniques: Activity Assay, Western Blot, Control, Dilution Assay, Flow Cytometry, Staining

Journal: Oncogenesis

Article Title: P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker

doi: 10.1038/s41389-024-00541-2

Figure Lengend Snippet: A , B Cell proliferation assays for X01 ( A ) and 448 ( B ) GSCs treated with P4HB inhibitor securinine in a dose-dependent manner. Data are presented as means ± SD, n = 4, no significant (ns), * P < 0.05, ** P < 0.01, *** P < 0.001, t-test. C Kaplan–Meier survival curves of mice implanted with X01 GSCs treated with Control or securinine ( n = 6 in each group, 1 × 10 5 GSCs/mouse, 15 mg/kg, I.P. injection), P = 0.0089, log-rank test. D H&E staining of the whole brains of mice bearing orthotopic xenografts of X01 GSCs treated with securinine, scale bar, 50 μm. E Schematic diagram of the high level of intracellular P4HB secreted into blood vessels. F , G Measurement of the serum protein level of P4HB in GBM patients and healthy controls from Henan Provincial People’s Hospital and Chinese PLA General Hospital in Beijing. *** P < 0.001, t-test. H Comprehensive overview of P4HB roles in GSCs tumorigenesis and its secretion into blood vessels as a diagnostic biomarker in GBM patients.

Article Snippet: V-ZAD-FMK, Z-DEVE-FMK dimethyl fumarate, ferrostatin-1, and the recombinant P4HB protein were purchased from MedChemExpress.

Techniques: Control, Injection, Staining, Diagnostic Assay, Biomarker Discovery

Journal: The Journal of Clinical Investigation

Article Title: The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis

doi: 10.1172/JCI80319

Figure Lengend Snippet: (A) Primers used for PCR. (B) PCR products of tail DNA from wild-type mice and transgenic PDI(ss-oo) mice. PCR with primers C and D gave the expected 441-bp product (no product was seen from the wild-type allele, as this includes exons and is too large for PCR). PCR with primers A and B gave the predicted 305-bp product for the transgenic PDI(ss-oo) and the corresponding 530-bp product from the wild-type allele (includes both intron and exon elements). PCR products of β-actin served as control. (C) RT-PCR products of platelet mRNA from wild-type and transgenic PDI(ss-oo) mice. (D) Sequencing of cDNA prepared from platelet mRNA shows the 2 G-to-C substitutions in the C-terminal active site domain. The PCR product using primers C and D was subjected to sequencing using primer A. (E) Western blot of platelet lysate using a polyclonal rabbit anti-PDI antibody shows equal amounts of PDI, ERp57, and ERp72. The PLCγ2 loading controls for PDI are shown. Separate loading controls were run for ERp57 and ERp72, with similar amounts of protein found in each sample (data not shown). Results in B–E are each representative of 3 separate experiments. (F) Platelet counts in wild-type and transgenic PDI(ss-oo) mice; mean ± SEM, n = 12, t test. (G) Normal expression of αIIbβ3, GPIb, and GPVI in platelets from transgenic PDI(ss-oo) mice compared with wild-type mice; mean ± SEM, n = 10, t test.

Article Snippet: Additional materials included IRDye 800–conjugated goat anti-mouse IgG and IRDye 680–conjugated goat anti-rabbit IgG (LI-COR Bioscience); PVDF membrane (Millipore); α-thrombin and quercetin-3-rutinoside (Sigma-Aldrich); poly(I:C) (R&D Systems); eptifibatide (Shering Plough); and PDI (P4HB) mouse cDNA clone (MC200577) (OriGene Technologies).

Techniques: Transgenic Assay, Control, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Expressing

Journal: Cell death & disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas.

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: Fig. 1 Role of PDIA1 in oxidant generation by colon carcinoma cells with distinct levels of KRas activation. a KRas expression and activity in Caco2, HKE3, and HCT116 cells. Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH (n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological). Test t **p < 0.01, (n = 4); e–g ROS production after 72 h of PDIA1 silencing, measured by HPLC analysis of DHE oxidation products. DHE oxidation produces, among many others, two major products: 2-hidroxyethidium (EOH), which is representative of superoxide species and ethidium, representative of other oxidant species. Ctrl negative, si-RNA control; si-PDI, si-RNA against PDIA1 protein. Representative immunoblots of PDI silencing for each cell type Test t *p < 0.05; **p < 0.01. For Caco2 cells, n = 3; HKE3 cells, n = 4; HCT116 cells, n = 5

Article Snippet: For PDIA1 silencing, cells were transfected with 300 nmol of PDIA1 triplex si-RNA (from OriGene) or stealth RNAi medium Universal negative control (from Invitrogen).

Techniques: Activation Assay, Expressing, Activity Assay, Control, Western Blot, Software, Titration, Enzyme-linked Immunosorbent Assay

Journal: Cell death & disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas.

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: Fig. 3 Rac1 and regulation of PDIA1 – Nox1 axis. a Superoxide production after 72 h of PDIA1 silencing in HKE3 transfected with Rac1G12V, treated or not with 10 µM of NoxA1ds (Nox1 peptide inhibitor), measured by DHE oxidation detected by HPLC. **p < 0.01, ANOVA plus Tukey's multiple comparison test (n = 3). b Superoxide production after 72 h of PDIA1 silencing in HCT116 cells treated or not with 50 µM of W56 Rac1 peptide inhibitor. *p < 0.05, ANOVA plus Tukey's multiple comparison test (n = 4). c PDIA1 co-immunoprecipitation, IP: PDIA1 immunoprecipitation in Caco2, HKE3, and HCT116 cells, IgG: Immunoglobulin control, Input: 1% of total protein lysate, IB: immunoblot against PDIA1, KRas, Rac1, GAPDH, and tubulin (n = 2). EOH: 2-hidroxyethidiium relative superoxide production

Article Snippet: For PDIA1 silencing, cells were transfected with 300 nmol of PDIA1 triplex si-RNA (from OriGene) or stealth RNAi medium Universal negative control (from Invitrogen).

Techniques: Transfection, Comparison, Immunoprecipitation, Control, Western Blot

Journal: Cell death & disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas.

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: Fig. 4 Effects of PDIA1 silencing on spheroid growth and invasiveness. 3D cell proliferation assay: a Representative phase-contrast images of spheroid proliferation with or without PDIA1 silencing; pictures were taken at T0 and T48 h after spheroid formation on methylcellulose media. Scale bar, 500 µm. b Proliferation analysis: spheroid surface area was measured at T0 and T48 h, using ImageJ software. Spheroid growth was calculated as T48 h spheroid area/T0 spheroid area; between 17 and 27 spheroids were analyzed for each condition. Data are mean fold ± SD, **p < 0.01; ****p < 0.0001 vs. HKE3 scrmb, ANOVA plus Tukey's multiple comparison test. c Effect of PDIA1 silencing on cell invasion: representative phase-contrast images of spheroid invasion in 2D fibronectin matrix (10 µM); pictures were taken at T0 and T48 h after spheroids were laid down on matrix. Scale bar, 500 µm. d Spheroid 2D invasion analysis: total spheroid expansion was measured at T0 and T48 h using ImageJ software. Spheroid expansion was calculated as (T48 h total evasion area—T0 initial spheroid area)/T0 initial spheroid area; between 12 and 17 spheroids were analyzed for each condition. Data are mean ± SD, ****p < 0.0001 vs. HKE3 scrmb, ANOVA plus Tukey's multiple comparison test

Article Snippet: For PDIA1 silencing, cells were transfected with 300 nmol of PDIA1 triplex si-RNA (from OriGene) or stealth RNAi medium Universal negative control (from Invitrogen).

Techniques: Proliferation Assay, Software, Comparison

Journal: Cell death & disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas.

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: Fig. 5 Effects of PDIA1 silencing in signaling targets and EMT marker expression. PathScan array analysis. All array’s spots were quantified and analyzed using ImageJ software. PDIA1 silencing was checked by immunoblot analysis. Ctrl, si-RNA negative control; si-PDI, si-RNA against PDIA1; ERK1/2, Thr202/Tyr204 phosphorylation; Stat1, Tyr701 phosphorylation; Stat3, Tyr705 phosphorylation; Akt, Thr308 phosphorylation; Akt, Ser473 phosphorylation; AMPKa, Thr172 phosphorylation, s6 ribosomal protein, Ser235/236 phosphorylation; mTOR, Ser2448 phosphorylation; HSP27, Ser78 phosphorylation; Bad, Ser112 phosphorylation; p70 S6 kinase, Thr389 phosphorylation; PRAS40, Thr246 phosphorylation; p53, Ser15 phosphorylation; p38, Thr180/Tyr182 phosphorylation; SAPK/JNK, Thr183/Tyr185 phosphorylation; PARP, Asp214 cleavage; Caspase-3, Asp175 Cleavage; GSK-3β, Ser9 phosphorylation. b GSK-3β, Ser9 phosphorylation plot. c Stat3, Tyr705 phosphorylation. d Basal E-cadherin (E-cad) protein expression by immunoblot normalized for GAPDH (n = 3). e Immunoblot for E-cadherin before and after PDIA1 silencing in Caco2 cells (n = 2). f Immunoblot for E-cadherin before and after PDIA1 silencing in HKE3 cells.; ns: not significant, Student's t test (n = 3). g Immunoblot for E-cadherin before and after PDIA1 silencing in HCT116 cells *P < 0.05, Student's t test (n = 3). Immunoblot densities were quantified using Odyssey software

Article Snippet: For PDIA1 silencing, cells were transfected with 300 nmol of PDIA1 triplex si-RNA (from OriGene) or stealth RNAi medium Universal negative control (from Invitrogen).

Techniques: Marker, Expressing, Software, Western Blot, Negative Control, Phospho-proteomics

Journal: Cell death & disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas.

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: Fig. 6 Analysis of protein–protein interaction network and functional pathways associated with PDIA1–Nox1 axis and KRas. a Interaction map fashioned with String 10.5 program (http://string-db.org), Network nodes represent proteins of Nox1, PDIA1 (P4HB), RhoGDIα (ARHGDIA), RAC1, BRAF, KRAS, GSK3β (GSK3B), STAT3, and E-cadherin (CDH1). Different colored lines displays predicted functional links. Interactions experimentally determined appear in pink, interactions curated from databases in blue, co-expression was represented in black and text data mining in green. The analysis showed: nine nodes with an average node degree of 3.56; number of edges: 16; expected number of edges: 4; average local clustering coefficient: 0.333; PPI enrichment p-value: 2.02e−05. b Top 10 ranked enriched proteins KEGG pathway analysis. Bar graphs show the –log of FDR (False discovery rate). High values correlated to higher probabilities. c Cellular component Gene ontology (GO) analysis

Article Snippet: For PDIA1 silencing, cells were transfected with 300 nmol of PDIA1 triplex si-RNA (from OriGene) or stealth RNAi medium Universal negative control (from Invitrogen).

Techniques: Functional Assay, Expressing

Journal: Cell death & disease

Article Title: Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas.

doi: 10.1038/s41419-019-1402-y

Figure Lengend Snippet: Fig. 7 Model of PDIA1-associated regulation of Nox1 in cells with normal or overactivated KRas

Article Snippet: For PDIA1 silencing, cells were transfected with 300 nmol of PDIA1 triplex si-RNA (from OriGene) or stealth RNAi medium Universal negative control (from Invitrogen).

Techniques:

Journal: Angiogenesis

Article Title: Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis

doi: 10.1007/s10456-022-09852-7

Figure Lengend Snippet: PDIA1 is required for postnatal angiogenesis in vivo. (a) Retinal whole-mount staining with Isolectin B4 (IB4) antibody of P5 pups from WT (n = 7) or Pdia1+/− (n = 9) mice. Arrowheads show tip cell sprouting and filopodia. The images show whole retina structure, remodeling plexus (branching points), and angiogenic fronts (tip cells). The quantitative results show retina length from center, number of branching points, and number of tip cells and the percentage of vascular retinal area to total retinal area. Results were presented as mean ± SEM. *p < 0.05. (b) Growth factor-reduced Matrigel containing VEGF (50 ng/mL), bFGF (100 ng/mF), heparin (60 unit) was injected subcutaneously in WT (n = 4) or Pdia1+/− (n = 4) mice and then harvested for analysis after 3 days. The neovascularization in Matrigel was determined by staining with isolectin-B4 antibody. The images were representative from 3 to 4 mice and presented as mean ± SEM. *p < 0.05, vs WT. (c) Aortic ring sprouting assay was monitored on Matrigel for 5–7 days [upper panel; lenti-scramble shRNA (shCont) vs lenti-shPdia1 RNA. Lower panel; WT mice (n = 3–4) vs Pdia1+/− mice (n = 3–4)]. Numbers of sprouting branch from endothelial cells were counted and normalized by control shRNA or WT mice. The images were representative from at least three different experiments or different mice and presented as mean ± SEM. *p < 0.05, **p < 0.01 vs control or WT. ns, not significant

Article Snippet: Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used.

Techniques: In Vivo, Staining, Injection, shRNA

Journal: Angiogenesis

Article Title: Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis

doi: 10.1007/s10456-022-09852-7

Figure Lengend Snippet: Endothelial PDIA1 is required for postnatal reparative angiogenesis in vivo. a and b, PDIA1 expression in ischemic and non-ischemic muscles at day 14 post-surgery by showing immunohisto-chemical analysis (a) and immunoblotting (b). (c) CD31 (red, EC marker) or PDIA1 (green) staining or their colocalization (merged, white arrows) in ischemic and non-ischemic gastrocnemius muscles at day 7 post-surgery. Scale bars, 20 μm. Bar graph represents CD31–PDIA1 co-localized cell numbers per field (n = 3, **p < 0.01). (d) Blood flow recovery as determined by the ratio of foot perfusion between ischemic (left) and ischemic (right) legs after hindlimb ischemia in WT and Pdia1ECKO mice (n = 5). Upper panels show representative laser Doppler flow images showing limb perfusion recovery (red arrows show ischemic foot) at day 28 after ischemia in WT and Pdia1ECKO mice, (e) Capillary density (CD31 staining) in ischemic and non-ischemic muscles of WT and Pdia1ECKO mice at day 21. Scale bars, 5 μm. Bottom panels show their quantitative analysis (number of capillaries per mm square). Data are mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used.

Techniques: In Vivo, Expressing, Western Blot, Marker, Staining

Journal: Angiogenesis

Article Title: Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis

doi: 10.1007/s10456-022-09852-7

Figure Lengend Snippet: PDIA1 is required for VEGF-induced signaling and angiogenesis in ECs. HUVECs transfected with control or PDIA1 siRNAs were stimulated with VEGF (20 ng/ml) for 6 h were and then used for EC migration as measured by modified Boyden chamber assay (n = 3) (a), EC proliferation as measured by BrdU assay (n = 3) (b), and Spheroid sprouting assay (n = 3–4) (c). (d) HUVECs transfected with control or PDIA1 siRNAs were stimulated with VEGF (20 ng/ml) for indicated time. Lysates were immunoblotted (IB) with antibodies indicated. Actin is loading control. Graph represents the averaged fold change over the basal control. Statistics analysis was performed by comparing two groups at each timepoint. (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used.

Techniques: Transfection, Migration, Modification, Boyden Chamber Assay, BrdU Staining

Journal: Angiogenesis

Article Title: Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis

doi: 10.1007/s10456-022-09852-7

Figure Lengend Snippet: VEGF stimulation promotes PDIA1 binding to internalized VEGFR2 at early endosomes. (a) HUVECs stimulated with VEGF (20 ng/ml) were immunoprecipitated (IP) with VEGFR2 antibody or IgG (negative control), followed by IB with antibodies indicated. Lower panel represents the averaged fold change of PDIA1/VEGFR2 ratio over the basal ratio (n = 3, *p < 0.05, **p < 0.01). (b) HUVECs stimulated with or without VEGF for 5 min and co-stained for PDIA1 with VEGFR2 or early endosome markers (Rab5) or late endosome marker (Rab7). Images were taken using confocal microscopy. Yellow fluorescence (white arrowheads) in merged images shows their co-localization (white box), which was analyzed by comparing the fluorescence intensity for each protein on white line in enlarged images (b1). Scale bars, 5μm. Bar graph represents the percentage of their co-localization (b2). Data are mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used.

Techniques: Binding Assay, Immunoprecipitation, Negative Control, Staining, Marker, Confocal Microscopy, Fluorescence

Journal: Angiogenesis

Article Title: Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis

doi: 10.1007/s10456-022-09852-7

Figure Lengend Snippet: VEGF induces sulfenylation of PDIA1 in a NOX4- and mitochondrial ROS-dependent manner in ECs. (a) HUVECs transfected with control or PDIA1 siRNAs stimulated with or without VEGF (20 ng/ml) for 5 min were used to measure dichlorofluorescein (DCF) fluorescence and DAPI staining (blue). Bottom panel represents the averaged fold change of fluorescence intensity over the basal control (n = 3). (b–d) DCP–Bio1 assay to detect PDIA1 sulfenylation (CysOH formation). Scheme of DCP–Bio1 assay to detect of sulfenylated protein (b, left panel). HUVECs were stimulated with VEGF for indicated time (b, right panel) or infected with Ad.null (cont), Ad.shNox4, or Ad.Mito-catalase (c), or Ad.flag–hPDIA1–WT or Ad.flag–hPDIA1–CS (redox-dead mutant) (d) and then stimulated with VEGF for 30 min. DCP–Bio1-labeled lysates were pulled down with streptavidin beads and then immunoblotted (IB) with anti-PDIA1 antibody to measure PDIA1–CysOH formation. Total lysates were IB with antibodies indicated. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01

Article Snippet: Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used.

Techniques: Transfection, Fluorescence, Staining, Infection, Mutagenesis, Labeling

Journal: Angiogenesis

Article Title: Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis

doi: 10.1007/s10456-022-09852-7

Figure Lengend Snippet: Sulfenylation of PDIA1 promotes VEGFR2 signaling and angiogenesis. (a, b, c, d) HUVECs were infected with Ad.null (Cont), Ad.PDIA1–WT or flag-hPDIA1–C/S (redox-dead mutant). Cells were stimulated with or without VEGF (20 ng/ml) for 5 min (a) or indicated times (b), and then lysates were IB with antibodies indicated (a and b). In (b), statistics analysis was performed by comparing two groups at each timepoint. In parallel, cells were used to measure EC migration with and without VEGF for 6 h, as measured by the modified Boyden chamber assay (c), and EC proliferation with and without VEGF for 24 h, as measured by BrdU assay (d). Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used.

Techniques: Infection, Mutagenesis, Migration, Modification, Boyden Chamber Assay, BrdU Staining

Journal: Angiogenesis

Article Title: Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis

doi: 10.1007/s10456-022-09852-7

Figure Lengend Snippet: PDIA1–CysOH promotes VEGFR2 signaling via oxidative inactivation of PTP1B. (a) HUVECs infected with Ad.null (Cont) or Ad.PTP1B–WT were stimulated with or without VEGF (20 ng/ml) for 5 min, and then lysates were immunoblotted with anti-p-VEGFR2 (Y1175) or antibodies indicated. (b) HUVECs stimulated with VEGF for indicated times were immunoprecipitated (IP) with PTP1B antibody or IgG (negative control), followed by IB with antibodies indicated. Lower panel represents the averaged fold change of PTP1B/PDIA1 ratio over the basal ratio (n = 3). (c) HUVECs infected with Ad.null or Ad. hPDIA1–CS (redox-dead mutant) were stimulated with VEGF for 5 min. DCP–Bio1-labeled lysates were pulled down with streptavidin beads and then IB with anti-PTP1B antibody to measure PTP1B–CysOH formation. Total lysates were IB with antibodies indicated. Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used.

Techniques: Infection, Immunoprecipitation, Negative Control, Mutagenesis, Labeling

Journal: Angiogenesis

Article Title: Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis

doi: 10.1007/s10456-022-09852-7

Figure Lengend Snippet: Proposed model. PDIA1 functions as a redox sensor that transmits VEGF-induced NOX–mitoROS signal via sulfenylation to promote VEGFR2 signaling and angiogenesis

Article Snippet: Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used.

Techniques: