Review



anti pdia1  (Cell Signaling Technology Inc)


Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96

    Structured Review

    Cell Signaling Technology Inc anti pdia1
    Anti Pdia1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 395 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/PDI+Rabbit+mAb/pmc12183083-222-28-29
    Average 96 stars, based on 395 article reviews
    anti pdia1 - by Bioz Stars, 2026-09
    96/100 stars

    Images

    Related Articles

    shRNA:

    Article Title: Fabrication and characterization of soy protein isolation-ferulic acid antioxidant hydrogels.
    Article Snippet: The NLRP3 inflammasome is a cytosolic protein complex that regulates innate immune signaling in response to diverse pathogenic insults through the proteolytic processing and secretion of pro-inflammatory cytokines such as IL1β.. Hyperactivation of NLRP3 inflammasome signaling is implicated in the onset and pathogenesis of numerous diseases, motivating the discovery of new strategies to suppress NLRP3 inflammasome activity.. We sought to define the potential for the proteostasis regulator AA147 to inhibit the assembly and activation of the NLRP3 inflammasome.

    Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation
    Article Snippet: G; Addgene cat. 12259). .. Mission shRNA plasmids in pLKO.1 vectors were obtained from La Jolla Institute for Allergy and Immunology (LJI) and were previously used in Paxman et al.38 The specific target sequences for viral plasmid of these shRNAs are below: The following primary antibodies were diluted as noted in 5 % bovine serum albumin (BSA) in TBS and incubated overnight: KDEL (1 : 1000; Enzo cat. ADI-SPA-827-F), NLRP3 (1 : 1000; Cell Signaling cat. 15101), CASP1 (1 : 1000; Cell Signaling cat. 3866), ASC (1 : 1000, Cell Signaling cat. 13833), IL-1β (1 : 1000, GeneTex cat. GTX74034), PDIA1 (1 : 1000, Cell Signaling cat. 45596), Tubulin (1 : 2000; Sigma-Aldrich T6074), GAPDH (1 : 1000, GeneTex cat. GTX627408), NEK7 (1 : 1000, Cell Signaling cat. 3057). ..

    Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation
    Article Snippet: G; Addgene cat. 12259). .. Mission shRNA plasmids in pLKO.1 vectors were obtained from La Jolla Institute for Allergy and Immunology (LJI) and were previously used in Paxman et al.38 The specific target sequences for viral plasmid of these shRNAs are below: Target (Designation) shRNA sequence The following primary antibodies were diluted as noted in 5% bovine serum albumin (BSA) in TBS and incubated overnight: KDEL (1:1000; Enzo cat. ADI-SPA-827-F), NLRP3 (1:1000; Cell Signaling cat. 15101), CASP1 (1:1000; Cell Signaling cat. 3866), ASC (1:1000, Cell Signaling cat. 13833), IL-1β (1:1000, GeneTex cat. GTX74034), PDIA1 (1:1000, Cell Signaling cat. 45596), Tubulin (1:2000; Sigma-Aldrich T6074), GAPDH (1:1000, GeneTex cat. GTX627408), NEK7 (1:1000, Cell Signaling cat. 3057). ..

    Plasmid Preparation:

    Article Title: Fabrication and characterization of soy protein isolation-ferulic acid antioxidant hydrogels.
    Article Snippet: The NLRP3 inflammasome is a cytosolic protein complex that regulates innate immune signaling in response to diverse pathogenic insults through the proteolytic processing and secretion of pro-inflammatory cytokines such as IL1β.. Hyperactivation of NLRP3 inflammasome signaling is implicated in the onset and pathogenesis of numerous diseases, motivating the discovery of new strategies to suppress NLRP3 inflammasome activity.. We sought to define the potential for the proteostasis regulator AA147 to inhibit the assembly and activation of the NLRP3 inflammasome.

    Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation
    Article Snippet: G; Addgene cat. 12259). .. Mission shRNA plasmids in pLKO.1 vectors were obtained from La Jolla Institute for Allergy and Immunology (LJI) and were previously used in Paxman et al.38 The specific target sequences for viral plasmid of these shRNAs are below: The following primary antibodies were diluted as noted in 5 % bovine serum albumin (BSA) in TBS and incubated overnight: KDEL (1 : 1000; Enzo cat. ADI-SPA-827-F), NLRP3 (1 : 1000; Cell Signaling cat. 15101), CASP1 (1 : 1000; Cell Signaling cat. 3866), ASC (1 : 1000, Cell Signaling cat. 13833), IL-1β (1 : 1000, GeneTex cat. GTX74034), PDIA1 (1 : 1000, Cell Signaling cat. 45596), Tubulin (1 : 2000; Sigma-Aldrich T6074), GAPDH (1 : 1000, GeneTex cat. GTX627408), NEK7 (1 : 1000, Cell Signaling cat. 3057). ..

    Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation
    Article Snippet: G; Addgene cat. 12259). .. Mission shRNA plasmids in pLKO.1 vectors were obtained from La Jolla Institute for Allergy and Immunology (LJI) and were previously used in Paxman et al.38 The specific target sequences for viral plasmid of these shRNAs are below: Target (Designation) shRNA sequence The following primary antibodies were diluted as noted in 5% bovine serum albumin (BSA) in TBS and incubated overnight: KDEL (1:1000; Enzo cat. ADI-SPA-827-F), NLRP3 (1:1000; Cell Signaling cat. 15101), CASP1 (1:1000; Cell Signaling cat. 3866), ASC (1:1000, Cell Signaling cat. 13833), IL-1β (1:1000, GeneTex cat. GTX74034), PDIA1 (1:1000, Cell Signaling cat. 45596), Tubulin (1:2000; Sigma-Aldrich T6074), GAPDH (1:1000, GeneTex cat. GTX627408), NEK7 (1:1000, Cell Signaling cat. 3057). ..

    Incubation:

    Article Title: Fabrication and characterization of soy protein isolation-ferulic acid antioxidant hydrogels.
    Article Snippet: The NLRP3 inflammasome is a cytosolic protein complex that regulates innate immune signaling in response to diverse pathogenic insults through the proteolytic processing and secretion of pro-inflammatory cytokines such as IL1β.. Hyperactivation of NLRP3 inflammasome signaling is implicated in the onset and pathogenesis of numerous diseases, motivating the discovery of new strategies to suppress NLRP3 inflammasome activity.. We sought to define the potential for the proteostasis regulator AA147 to inhibit the assembly and activation of the NLRP3 inflammasome.

    Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation
    Article Snippet: G; Addgene cat. 12259). .. Mission shRNA plasmids in pLKO.1 vectors were obtained from La Jolla Institute for Allergy and Immunology (LJI) and were previously used in Paxman et al.38 The specific target sequences for viral plasmid of these shRNAs are below: The following primary antibodies were diluted as noted in 5 % bovine serum albumin (BSA) in TBS and incubated overnight: KDEL (1 : 1000; Enzo cat. ADI-SPA-827-F), NLRP3 (1 : 1000; Cell Signaling cat. 15101), CASP1 (1 : 1000; Cell Signaling cat. 3866), ASC (1 : 1000, Cell Signaling cat. 13833), IL-1β (1 : 1000, GeneTex cat. GTX74034), PDIA1 (1 : 1000, Cell Signaling cat. 45596), Tubulin (1 : 2000; Sigma-Aldrich T6074), GAPDH (1 : 1000, GeneTex cat. GTX627408), NEK7 (1 : 1000, Cell Signaling cat. 3057). ..

    Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation
    Article Snippet: G; Addgene cat. 12259). .. Mission shRNA plasmids in pLKO.1 vectors were obtained from La Jolla Institute for Allergy and Immunology (LJI) and were previously used in Paxman et al.38 The specific target sequences for viral plasmid of these shRNAs are below: Target (Designation) shRNA sequence The following primary antibodies were diluted as noted in 5% bovine serum albumin (BSA) in TBS and incubated overnight: KDEL (1:1000; Enzo cat. ADI-SPA-827-F), NLRP3 (1:1000; Cell Signaling cat. 15101), CASP1 (1:1000; Cell Signaling cat. 3866), ASC (1:1000, Cell Signaling cat. 13833), IL-1β (1:1000, GeneTex cat. GTX74034), PDIA1 (1:1000, Cell Signaling cat. 45596), Tubulin (1:2000; Sigma-Aldrich T6074), GAPDH (1:1000, GeneTex cat. GTX627408), NEK7 (1:1000, Cell Signaling cat. 3057). ..

    Sequencing:

    Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation
    Article Snippet: G; Addgene cat. 12259). .. Mission shRNA plasmids in pLKO.1 vectors were obtained from La Jolla Institute for Allergy and Immunology (LJI) and were previously used in Paxman et al.38 The specific target sequences for viral plasmid of these shRNAs are below: Target (Designation) shRNA sequence The following primary antibodies were diluted as noted in 5% bovine serum albumin (BSA) in TBS and incubated overnight: KDEL (1:1000; Enzo cat. ADI-SPA-827-F), NLRP3 (1:1000; Cell Signaling cat. 15101), CASP1 (1:1000; Cell Signaling cat. 3866), ASC (1:1000, Cell Signaling cat. 13833), IL-1β (1:1000, GeneTex cat. GTX74034), PDIA1 (1:1000, Cell Signaling cat. 45596), Tubulin (1:2000; Sigma-Aldrich T6074), GAPDH (1:1000, GeneTex cat. GTX627408), NEK7 (1:1000, Cell Signaling cat. 3057). ..



    Similar Products

    90
    ProSpec recombinant pdia1
    Recombinant Pdia1, supplied by ProSpec, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/pdia1++enz+262/pm40216967-186-18-23
    Average 90 stars, based on 1 article reviews
    recombinant pdia1 - by Bioz Stars, 2026-09
    90/100 stars
      Buy from Supplier

    90
    Thermo Fisher anti‐pdia1 (rabbit)
    Anti‐Pdia1 (Rabbit), supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/mouse+pdia1+detection+antibody/pm40178024-126-7-11
    Average 90 stars, based on 1 article reviews
    anti‐pdia1 (rabbit) - by Bioz Stars, 2026-09
    90/100 stars
      Buy from Supplier

    90
    Human Protein Atlas pdia1 protein
    Endoplasmic reticulum stress induces <t>PDIA1</t> retention and precedes mitochondrial dysfunction following exposure to lethal doses of CSE. (A) MLE12 cells were treated with 20 % CSE for 15 min and analyzed for changes in stress signaling proteins. Actin was used as a loading control. (B) Cell viability following exposure to 20 % CSE for the indicated times was assessed using the CellTiterGlo assay in at least three independent experiments. (C–F) MLE12 cells were exposed to 20 % CSE for 15 min. (C) PDIA1 and mitochondria were visualized, with the left images showing 3D reconstructions of the entire cell. ( D )The graph quantifies the relative volumes of nuclei, ER, and mitochondria in cells, analyzed using the 3D suite function of FUJI ImageJ software. Data is from 8 to 10 cells per condition from four independent experiments. (E) Total (T), membrane (M), and cytoplasmic (CP) protein fractions were isolated and analyzed for PDIA1 levels. (F) ER-enriched microsomes (ER 12,000), smaller membranes and vesicles (M 30,000), and cytosolic pellets (P 54,000) were isolated and analyzed for PDIA1 levels. All data is representative of at least three independent experiments.
    Pdia1 Protein, supplied by Human Protein Atlas, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/pdia4+protein/pmc12172305-224-34-40
    Average 90 stars, based on 1 article reviews
    pdia1 protein - by Bioz Stars, 2026-09
    90/100 stars
      Buy from Supplier

    94
    Proteintech anti pdia1
    Endoplasmic reticulum stress induces <t>PDIA1</t> retention and precedes mitochondrial dysfunction following exposure to lethal doses of CSE. (A) MLE12 cells were treated with 20 % CSE for 15 min and analyzed for changes in stress signaling proteins. Actin was used as a loading control. (B) Cell viability following exposure to 20 % CSE for the indicated times was assessed using the CellTiterGlo assay in at least three independent experiments. (C–F) MLE12 cells were exposed to 20 % CSE for 15 min. (C) PDIA1 and mitochondria were visualized, with the left images showing 3D reconstructions of the entire cell. ( D )The graph quantifies the relative volumes of nuclei, ER, and mitochondria in cells, analyzed using the 3D suite function of FUJI ImageJ software. Data is from 8 to 10 cells per condition from four independent experiments. (E) Total (T), membrane (M), and cytoplasmic (CP) protein fractions were isolated and analyzed for PDIA1 levels. (F) ER-enriched microsomes (ER 12,000), smaller membranes and vesicles (M 30,000), and cytosolic pellets (P 54,000) were isolated and analyzed for PDIA1 levels. All data is representative of at least three independent experiments.
    Anti Pdia1, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/PDI+Antibody/pm40645933-277-69-70
    Average 94 stars, based on 1 article reviews
    anti pdia1 - by Bioz Stars, 2026-09
    94/100 stars
      Buy from Supplier

    90
    abberior instruments pdia1 abberior star-488
    Endoplasmic reticulum stress induces <t>PDIA1</t> retention and precedes mitochondrial dysfunction following exposure to lethal doses of CSE. (A) MLE12 cells were treated with 20 % CSE for 15 min and analyzed for changes in stress signaling proteins. Actin was used as a loading control. (B) Cell viability following exposure to 20 % CSE for the indicated times was assessed using the CellTiterGlo assay in at least three independent experiments. (C–F) MLE12 cells were exposed to 20 % CSE for 15 min. (C) PDIA1 and mitochondria were visualized, with the left images showing 3D reconstructions of the entire cell. ( D )The graph quantifies the relative volumes of nuclei, ER, and mitochondria in cells, analyzed using the 3D suite function of FUJI ImageJ software. Data is from 8 to 10 cells per condition from four independent experiments. (E) Total (T), membrane (M), and cytoplasmic (CP) protein fractions were isolated and analyzed for PDIA1 levels. (F) ER-enriched microsomes (ER 12,000), smaller membranes and vesicles (M 30,000), and cytosolic pellets (P 54,000) were isolated and analyzed for PDIA1 levels. All data is representative of at least three independent experiments.
    Pdia1 Abberior Star 488, supplied by abberior instruments, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/abberior+star+488/pm40472774-232-0-1
    Average 90 stars, based on 1 article reviews
    pdia1 abberior star-488 - by Bioz Stars, 2026-09
    90/100 stars
      Buy from Supplier

    96
    Cell Signaling Technology Inc anti pdia1
    Endoplasmic reticulum stress induces <t>PDIA1</t> retention and precedes mitochondrial dysfunction following exposure to lethal doses of CSE. (A) MLE12 cells were treated with 20 % CSE for 15 min and analyzed for changes in stress signaling proteins. Actin was used as a loading control. (B) Cell viability following exposure to 20 % CSE for the indicated times was assessed using the CellTiterGlo assay in at least three independent experiments. (C–F) MLE12 cells were exposed to 20 % CSE for 15 min. (C) PDIA1 and mitochondria were visualized, with the left images showing 3D reconstructions of the entire cell. ( D )The graph quantifies the relative volumes of nuclei, ER, and mitochondria in cells, analyzed using the 3D suite function of FUJI ImageJ software. Data is from 8 to 10 cells per condition from four independent experiments. (E) Total (T), membrane (M), and cytoplasmic (CP) protein fractions were isolated and analyzed for PDIA1 levels. (F) ER-enriched microsomes (ER 12,000), smaller membranes and vesicles (M 30,000), and cytosolic pellets (P 54,000) were isolated and analyzed for PDIA1 levels. All data is representative of at least three independent experiments.
    Anti Pdia1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/PDI+Rabbit+mAb/pmc12183083-222-28-29
    Average 96 stars, based on 1 article reviews
    anti pdia1 - by Bioz Stars, 2026-09
    96/100 stars
      Buy from Supplier

    90
    Cell Signaling Technology Inc pdia1 (p4hb) antibody no. 2466
    Endoplasmic reticulum stress induces <t>PDIA1</t> retention and precedes mitochondrial dysfunction following exposure to lethal doses of CSE. (A) MLE12 cells were treated with 20 % CSE for 15 min and analyzed for changes in stress signaling proteins. Actin was used as a loading control. (B) Cell viability following exposure to 20 % CSE for the indicated times was assessed using the CellTiterGlo assay in at least three independent experiments. (C–F) MLE12 cells were exposed to 20 % CSE for 15 min. (C) PDIA1 and mitochondria were visualized, with the left images showing 3D reconstructions of the entire cell. ( D )The graph quantifies the relative volumes of nuclei, ER, and mitochondria in cells, analyzed using the 3D suite function of FUJI ImageJ software. Data is from 8 to 10 cells per condition from four independent experiments. (E) Total (T), membrane (M), and cytoplasmic (CP) protein fractions were isolated and analyzed for PDIA1 levels. (F) ER-enriched microsomes (ER 12,000), smaller membranes and vesicles (M 30,000), and cytosolic pellets (P 54,000) were isolated and analyzed for PDIA1 levels. All data is representative of at least three independent experiments.
    Pdia1 (P4hb) Antibody No. 2466, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/antibodies+against+pdia1+cell+signaling+3501s/pm39809267-1356-0-13
    Average 90 stars, based on 1 article reviews
    pdia1 (p4hb) antibody no. 2466 - by Bioz Stars, 2026-09
    90/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc pdia1 antibody cell signaling technology
    Figure 3. Natural source phenethyl isothio- cyanate improves AAT-Z monomer secre- tion and NE inhibitory activity (A) The chemical structure of phenethyl isothio- cyanate (PEITC). (B) Immunoblotting analysis of extracellular AAT-Z in the absence or presence of PEITC in lung cells. IB3null-Z cells were treated with PEITC (5 mM) for 24 h; the cell lysate and culture medium were collected for SDS-PAGE and immunoblotting to measure AAT-Z total secretion. AAT-WT expres- sion was used as control. (C and D) AAT-Z monomer secretion (C) and NE inhibitory activity (D) in the absence or presence of PEITC (5 mM) for 24 h in IB3null-Z cells were measured by using an ELISA assay and NE inhibitory fluorogenic assay, respectively. (E and F) AAT-Z monomer secretion (E) and NE inhibitory activity (F) in the absence or presence of PEITC from Huh7.5null-Z were measured by ELISA and NE inhibitory fluorogenic assays, respectively. (G and H) The impact of PEITC in AAT-Z HLCs. Cells were treated by PEITC with 5, 10, or 20 mM for 24 h. The AAT-Z secreted monomer (G) and NE inhibitory activity (H) in the absence or presence of PEITC were measured by ELSIA assay and NE inhibitory fluorogenic assay. (I and J) A PDI general inhibitor eliminates PEITC correction of AAT-Z. IB3null-Z cells expressing AAT-Z were treated with the PDI inhibitor 16F16 (20 mM) in the absence or presence of PEITC (5 mM) for 24 h. Cell culture medium was collected, and AAT-Z secreted monomer (I) and NE inhibitory activity measured (J). (K and L) PDIA4 silencing eliminates PEITC correction of AAT-Z secretion and NE inhibitory activity. <t>PDIA1,</t> PDIA3, or PDIA4 expression were silenced by siRNA transfection in IB3null-Z
    Pdia1 Antibody Cell Signaling Technology, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pdia1/SUZ12+XP+Rabbit+mAb/pm39809267-1332-41-43
    Average 94 stars, based on 1 article reviews
    pdia1 antibody cell signaling technology - by Bioz Stars, 2026-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    Endoplasmic reticulum stress induces PDIA1 retention and precedes mitochondrial dysfunction following exposure to lethal doses of CSE. (A) MLE12 cells were treated with 20 % CSE for 15 min and analyzed for changes in stress signaling proteins. Actin was used as a loading control. (B) Cell viability following exposure to 20 % CSE for the indicated times was assessed using the CellTiterGlo assay in at least three independent experiments. (C–F) MLE12 cells were exposed to 20 % CSE for 15 min. (C) PDIA1 and mitochondria were visualized, with the left images showing 3D reconstructions of the entire cell. ( D )The graph quantifies the relative volumes of nuclei, ER, and mitochondria in cells, analyzed using the 3D suite function of FUJI ImageJ software. Data is from 8 to 10 cells per condition from four independent experiments. (E) Total (T), membrane (M), and cytoplasmic (CP) protein fractions were isolated and analyzed for PDIA1 levels. (F) ER-enriched microsomes (ER 12,000), smaller membranes and vesicles (M 30,000), and cytosolic pellets (P 54,000) were isolated and analyzed for PDIA1 levels. All data is representative of at least three independent experiments.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: Endoplasmic reticulum stress induces PDIA1 retention and precedes mitochondrial dysfunction following exposure to lethal doses of CSE. (A) MLE12 cells were treated with 20 % CSE for 15 min and analyzed for changes in stress signaling proteins. Actin was used as a loading control. (B) Cell viability following exposure to 20 % CSE for the indicated times was assessed using the CellTiterGlo assay in at least three independent experiments. (C–F) MLE12 cells were exposed to 20 % CSE for 15 min. (C) PDIA1 and mitochondria were visualized, with the left images showing 3D reconstructions of the entire cell. ( D )The graph quantifies the relative volumes of nuclei, ER, and mitochondria in cells, analyzed using the 3D suite function of FUJI ImageJ software. Data is from 8 to 10 cells per condition from four independent experiments. (E) Total (T), membrane (M), and cytoplasmic (CP) protein fractions were isolated and analyzed for PDIA1 levels. (F) ER-enriched microsomes (ER 12,000), smaller membranes and vesicles (M 30,000), and cytosolic pellets (P 54,000) were isolated and analyzed for PDIA1 levels. All data is representative of at least three independent experiments.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Control, Software, Membrane, Isolation

    Proximity of PDIA1 to mitochondria increases following exposure to lethal doses of CSE. MLE12 cells were exposed to 20 % CSE for 15 min. (A) Left panels show merged images of MLE12 cells exposed (lower) or not exposed (upper) to CSE. A single plane showing the maximal intensity of both signals is displayed. Enlarged images of the boxed areas are shown on the right, with white arrows indicating mitochondrial-PDIA1 colocalization. (B) Enlarged images from (A, left panel) with line scans drawn across the mitochondria and PDIA1. Relative fluorescence intensities of each channel in each pixel are displayed as graphs on the left. Black arrows indicate areas where both fluorescence intensities are high. Scale bar = 125 nm. (C) Pearson's coefficient was calculated from boxed areas shown in (A) using the Coloc2 function in FUJI ImageJ software. Data represent a representative experiment from six independent replicates, with 3–5 areas analyzed per condition in each experiment.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: Proximity of PDIA1 to mitochondria increases following exposure to lethal doses of CSE. MLE12 cells were exposed to 20 % CSE for 15 min. (A) Left panels show merged images of MLE12 cells exposed (lower) or not exposed (upper) to CSE. A single plane showing the maximal intensity of both signals is displayed. Enlarged images of the boxed areas are shown on the right, with white arrows indicating mitochondrial-PDIA1 colocalization. (B) Enlarged images from (A, left panel) with line scans drawn across the mitochondria and PDIA1. Relative fluorescence intensities of each channel in each pixel are displayed as graphs on the left. Black arrows indicate areas where both fluorescence intensities are high. Scale bar = 125 nm. (C) Pearson's coefficient was calculated from boxed areas shown in (A) using the Coloc2 function in FUJI ImageJ software. Data represent a representative experiment from six independent replicates, with 3–5 areas analyzed per condition in each experiment.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Fluorescence, Software

    CSE induces S-glutathionylation of PDIA1. (A, B) Recombinant PDI proteins were treated by 20 % CSE and 5 mM GSH at 37 °C for 30 min, part of each sample was separated on a non-reducing gel and probed for the presence of S-glutathionylation (A), the other part was desalted by Biospin 6 columns. The protein was digested with endoproteinase LysC, the digest desalted, and the peptides subjected to LC-MS/MS identification (B). Spectra of domain a (left) and a’ (right) are shown. C fragment ions (from the peptide N-terminal) obtained by electron transfer dissociation (ETD) are shown in blue; while Z fragments (from the peptide C-terminal) are shown in red. The glutathione diagnostic fragment is shown in green. The glutathionylated cysteines are shown with an ∗ (C) A docked model of PDIA1 WT and PDIA1 FLFL with GSH, based on the human PDIA1 domain a’ (PDB ID: 3UEM ), showing disrupted interactions in the mutant version. His399 and Lys401 of PDIA1 WT interact with GSH, but these interactions are lost in PDIA1 FLFL , where His399 and Lys401 are replaced by Phe and Leu, respectively. (D) Fluorescent FRET-based analysis showing PDIA1 and GSTP1 interaction. The Y-axis represents the decrease in Alexa546 emission, indicating GSTP1 binding to PDIA1 WT or PDIA1 FLFL . The X-axis represents the total amount of GSTP1 labeled with QSY35. Data represent three independent experiments. (E) PDIA1 was immunoprecipitated from MLE12 cells, and GSTP1 was detected as co-immunoprecipitating with PDIA1 in three independent experiments. ( F ) Docked models of PDIA1 WT and PDIA1 FLFL with GSTP1, based on the human PDIA1 (PDB ID: 3UEM ) and GSTP1 (PDB ID: 6GSS ). GSH in this model is adapted from the 6GSS structure. PDIA1 WT Lys401 interacts with GSTP1 Gln51, and His399 is close to GSTP1 Asp94. These interactions are disrupted in PDIA1 FLFL , where His399 and Lys401 are replaced by Phe and Leu. ( G-H ) The model structure of wild type versus H399F, K401L PDIA1 and their residue-specific interaction map with GSTP1 build using Ligplot software. Please note that changes in the interaction map of His399Phe and Lys401Leu (PDIA1 WT → PDIA1 FLFL ) weakens interaction with GSTP1 and in GSH repositioning. The direct ionic interactions between PDIA1-His399 and GSTP1-Asp94, and PDIA1-Lys401 and GSTP1-Gln51 do not occur in PDIA1 FLFL . Moreover, the carboxyl oxygen of PDIA1-Trp396 interacts with GSTP1-Gln64 in PDIA1 WT and is replaced by an interaction with the side chain of GSTP1-Arg13 in the case of PDIA1 FLFL , compromising interactive interphase between those protein. Direct interaction of PDIA1-Lys401 with oxygen of GSH is abolished as well.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: CSE induces S-glutathionylation of PDIA1. (A, B) Recombinant PDI proteins were treated by 20 % CSE and 5 mM GSH at 37 °C for 30 min, part of each sample was separated on a non-reducing gel and probed for the presence of S-glutathionylation (A), the other part was desalted by Biospin 6 columns. The protein was digested with endoproteinase LysC, the digest desalted, and the peptides subjected to LC-MS/MS identification (B). Spectra of domain a (left) and a’ (right) are shown. C fragment ions (from the peptide N-terminal) obtained by electron transfer dissociation (ETD) are shown in blue; while Z fragments (from the peptide C-terminal) are shown in red. The glutathione diagnostic fragment is shown in green. The glutathionylated cysteines are shown with an ∗ (C) A docked model of PDIA1 WT and PDIA1 FLFL with GSH, based on the human PDIA1 domain a’ (PDB ID: 3UEM ), showing disrupted interactions in the mutant version. His399 and Lys401 of PDIA1 WT interact with GSH, but these interactions are lost in PDIA1 FLFL , where His399 and Lys401 are replaced by Phe and Leu, respectively. (D) Fluorescent FRET-based analysis showing PDIA1 and GSTP1 interaction. The Y-axis represents the decrease in Alexa546 emission, indicating GSTP1 binding to PDIA1 WT or PDIA1 FLFL . The X-axis represents the total amount of GSTP1 labeled with QSY35. Data represent three independent experiments. (E) PDIA1 was immunoprecipitated from MLE12 cells, and GSTP1 was detected as co-immunoprecipitating with PDIA1 in three independent experiments. ( F ) Docked models of PDIA1 WT and PDIA1 FLFL with GSTP1, based on the human PDIA1 (PDB ID: 3UEM ) and GSTP1 (PDB ID: 6GSS ). GSH in this model is adapted from the 6GSS structure. PDIA1 WT Lys401 interacts with GSTP1 Gln51, and His399 is close to GSTP1 Asp94. These interactions are disrupted in PDIA1 FLFL , where His399 and Lys401 are replaced by Phe and Leu. ( G-H ) The model structure of wild type versus H399F, K401L PDIA1 and their residue-specific interaction map with GSTP1 build using Ligplot software. Please note that changes in the interaction map of His399Phe and Lys401Leu (PDIA1 WT → PDIA1 FLFL ) weakens interaction with GSTP1 and in GSH repositioning. The direct ionic interactions between PDIA1-His399 and GSTP1-Asp94, and PDIA1-Lys401 and GSTP1-Gln51 do not occur in PDIA1 FLFL . Moreover, the carboxyl oxygen of PDIA1-Trp396 interacts with GSTP1-Gln64 in PDIA1 WT and is replaced by an interaction with the side chain of GSTP1-Arg13 in the case of PDIA1 FLFL , compromising interactive interphase between those protein. Direct interaction of PDIA1-Lys401 with oxygen of GSH is abolished as well.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Recombinant, Liquid Chromatography with Mass Spectroscopy, Diagnostic Assay, Mutagenesis, Binding Assay, Labeling, Immunoprecipitation, Residue, Software

    S-glutathionylated PDIA1 relocates to the vicinity of mitochondria (ERMCS). MLE12 cells were loaded with Mito-view, exposed to 20 % CSE for 15 min, or not, as indicated. ( A ) PDIA1 was visualized with Alexa-488, mitochondria with Mito-view, and glutathione with Alexa-568; single planes are shown. Enlarged images from the boxed areas are displayed in the middle panels. Line scans of these images are shown in the right panels with relative fluorescence intensities of each channel in each pixel plotted. (B) Reconstituted images of S-glutathionylated PDIA1 (green) and mitochondria (red), with an overlay created using ImageJ software. (C) Reconstituted images of non-glutathionylated PDIA1 and its overlay with mitochondria. (D) Graph shows the relative volumes of overlap between S-glutathionylated PDIA1 and mitochondria compared to non-glutathionylated PDIA1 and mitochondria. Images are representative of three independent experiments. (E) MLE12 cells were exposed, or not, to 20 % CSE for 2 h, crude mitochondrial fractions were isolated, and S-glutathionylated proteins were immunoprecipitated. The IP was resolved on gels, and S-glutathionylated PDIA1 was analyzed. The mitochondrial marker VDAC was used as an input control. Data are representative of three independent experiments.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: S-glutathionylated PDIA1 relocates to the vicinity of mitochondria (ERMCS). MLE12 cells were loaded with Mito-view, exposed to 20 % CSE for 15 min, or not, as indicated. ( A ) PDIA1 was visualized with Alexa-488, mitochondria with Mito-view, and glutathione with Alexa-568; single planes are shown. Enlarged images from the boxed areas are displayed in the middle panels. Line scans of these images are shown in the right panels with relative fluorescence intensities of each channel in each pixel plotted. (B) Reconstituted images of S-glutathionylated PDIA1 (green) and mitochondria (red), with an overlay created using ImageJ software. (C) Reconstituted images of non-glutathionylated PDIA1 and its overlay with mitochondria. (D) Graph shows the relative volumes of overlap between S-glutathionylated PDIA1 and mitochondria compared to non-glutathionylated PDIA1 and mitochondria. Images are representative of three independent experiments. (E) MLE12 cells were exposed, or not, to 20 % CSE for 2 h, crude mitochondrial fractions were isolated, and S-glutathionylated proteins were immunoprecipitated. The IP was resolved on gels, and S-glutathionylated PDIA1 was analyzed. The mitochondrial marker VDAC was used as an input control. Data are representative of three independent experiments.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Fluorescence, Software, Isolation, Immunoprecipitation, Marker, Control

    Inhibiting GSTP1-mediated S-glutathionylation prevents PDIA1 relocation to ERMCS upon CSE exposure. MLE12 cells were treated as indicated: (i) treated with GSTP1 inhibitor TLK199, or (ii) transfected with siRNA-Control/siRNA-GSTP1 for 24 h. Prior to experiments, cells were loaded with Mito-tracker-red and exposed, or not, to 15 min of 20 % CSE. PDIA1 (Abberior STAR-488), mitochondria (Mito-tracker-red), and nuclei (Hoechst) were visualized. (A, H) Representative single-plane images. (B, I) Enlarged images from boxed areas in (A). Arrows indicate PDIA1-mitochondria co-localization in CSE-treated cells. (C, J) Further enlarged images from boxed areas in (B) with line scans crossing mitochondria and PDIA1. (D, K) Graph showing line scan data with relative fluorescence intensity per pixel. Arrows indicate co-localization regions. (E, L) Pearson's coefficient calculated from boxed areas in (A) using the Coloc2 function in FUJI ImageJ software. (F, M) Graph summarizing Pearson's coefficient from three independent experiments. Significant differences ( p < 0.001) between control and CSE-exposed cells with inhibited S-glutathionylation indicate partial but not complete rescue. Statistical comparisons are detailed in Sup. .

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: Inhibiting GSTP1-mediated S-glutathionylation prevents PDIA1 relocation to ERMCS upon CSE exposure. MLE12 cells were treated as indicated: (i) treated with GSTP1 inhibitor TLK199, or (ii) transfected with siRNA-Control/siRNA-GSTP1 for 24 h. Prior to experiments, cells were loaded with Mito-tracker-red and exposed, or not, to 15 min of 20 % CSE. PDIA1 (Abberior STAR-488), mitochondria (Mito-tracker-red), and nuclei (Hoechst) were visualized. (A, H) Representative single-plane images. (B, I) Enlarged images from boxed areas in (A). Arrows indicate PDIA1-mitochondria co-localization in CSE-treated cells. (C, J) Further enlarged images from boxed areas in (B) with line scans crossing mitochondria and PDIA1. (D, K) Graph showing line scan data with relative fluorescence intensity per pixel. Arrows indicate co-localization regions. (E, L) Pearson's coefficient calculated from boxed areas in (A) using the Coloc2 function in FUJI ImageJ software. (F, M) Graph summarizing Pearson's coefficient from three independent experiments. Significant differences ( p < 0.001) between control and CSE-exposed cells with inhibited S-glutathionylation indicate partial but not complete rescue. Statistical comparisons are detailed in Sup. .

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Transfection, Control, Fluorescence, Software

    IMC profiling of lung parenchyma reveals the impact of aging and cigarette smoke exposure on PDIA1, GSTP1, and mitochondrial bioenergetic markers in AEC2s. (A) Representative IMC images of 14-week-old non-smoker (NS) mouse lung parenchyma using a 10-marker panel. Shown are proSPC (164Dy; AEC2 marker), PDIA1 (170Er), GSTP1 (168Er), and nuclear DNA (191Ir and 193Ir). Yellow arrows indicate representative AEC2. Yellow arrows point to the representative AEC2. ( B, D-F ) Single-cell quantitative analysis of marker expression across conditions. ( B ) PDIA1 relative intensity in AEC1s and AEC2. Each dot represents a single cell. ( C) Schematic outlining the experimental timeline and cigarette smoke (CS) exposure protocol. C57Bl/6 mice of different ages were exposed or not exposed to CS for defined durations. ( D–E) Violin plots showing single-cell quantification of relative signal intensity per AEC2 for PDIA1 (D) and GSTP1 (E) across NS and smoker (SM) groups. ( F ) Quantification of AEC2 abundance, expressed as the percentage of AEC2s per region of interest (ROI; 500 × 500 μm), across experimental groups. Data are presented as mean ± standard deviation from three biological replicates (N = 3 mice per group), each with technical triplicates (3 Regions of Interests (ROIs) per mouse). Final statistical comparisons were performed using unpaired two-tailed t-tests. P-values <0.05 were considered statistically significant.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: IMC profiling of lung parenchyma reveals the impact of aging and cigarette smoke exposure on PDIA1, GSTP1, and mitochondrial bioenergetic markers in AEC2s. (A) Representative IMC images of 14-week-old non-smoker (NS) mouse lung parenchyma using a 10-marker panel. Shown are proSPC (164Dy; AEC2 marker), PDIA1 (170Er), GSTP1 (168Er), and nuclear DNA (191Ir and 193Ir). Yellow arrows indicate representative AEC2. Yellow arrows point to the representative AEC2. ( B, D-F ) Single-cell quantitative analysis of marker expression across conditions. ( B ) PDIA1 relative intensity in AEC1s and AEC2. Each dot represents a single cell. ( C) Schematic outlining the experimental timeline and cigarette smoke (CS) exposure protocol. C57Bl/6 mice of different ages were exposed or not exposed to CS for defined durations. ( D–E) Violin plots showing single-cell quantification of relative signal intensity per AEC2 for PDIA1 (D) and GSTP1 (E) across NS and smoker (SM) groups. ( F ) Quantification of AEC2 abundance, expressed as the percentage of AEC2s per region of interest (ROI; 500 × 500 μm), across experimental groups. Data are presented as mean ± standard deviation from three biological replicates (N = 3 mice per group), each with technical triplicates (3 Regions of Interests (ROIs) per mouse). Final statistical comparisons were performed using unpaired two-tailed t-tests. P-values <0.05 were considered statistically significant.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Marker, Expressing, Standard Deviation, Two Tailed Test

    Introduction of exogenous PDI FLFL variant changes the ratio of unmodified to S-glutathionylated PDIA1. MLE12 cells were transfected with either pcDNA3.1 PDI WT , or pcDNA3.1 PDI FLFL , 18–22 h post transfection cells were exposed or not to 20 % CSE for 20 min and fixed for analysis. Exogenous PDIA1 was visualize by antibody that recognizes FLAG epitope, mitochondria by mitoSox-Red, glutathione by specific to it antibodies. ( A, and D ) Cells expressing PDI WT ( A) and PDI FLFL (D) respectively exposed or not to CSE. ( B, and E ) Overlay of mitochondrial and un-glutathionylated PDIA1-FLAG masks of enlarged boxed areas from A and D. Single planes with maximal signal intensities in both channels (FLAG and mitoSox) are shown on the left, 3D reconstitution of the whole area on the right. ( C, and F ) Overlay of mitochondrial and glutathionylated PDIA1-FLAG masks of enlarged boxed areas from A and D. Same single planes as in B and E with maximal signal intensities in both channels are shown on the left, 3D reconstitution of the whole area on the right. ( G ) Quantification of un-glutathionylated and glutathionylated PDIA1-FLAG. ( H ) Quantification of associated with mitochondria un-glutathionylated and glutathionylated PDIA1-FLAG. ( I ) Western blot of transfected with PDI FLFL−FLAG cells. PDIA1 is visualized by anti-PDI antibodies. Upper band corresponds to PDI FLFL−FLAG , lower band to endogenous PDIA1. Ratio of PDI FLFL−FLAG /PDIA1 endogenous calculated via densitometry using ImageLab 6.1 (Biorad) Volume Tool with rectangular selection.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: Introduction of exogenous PDI FLFL variant changes the ratio of unmodified to S-glutathionylated PDIA1. MLE12 cells were transfected with either pcDNA3.1 PDI WT , or pcDNA3.1 PDI FLFL , 18–22 h post transfection cells were exposed or not to 20 % CSE for 20 min and fixed for analysis. Exogenous PDIA1 was visualize by antibody that recognizes FLAG epitope, mitochondria by mitoSox-Red, glutathione by specific to it antibodies. ( A, and D ) Cells expressing PDI WT ( A) and PDI FLFL (D) respectively exposed or not to CSE. ( B, and E ) Overlay of mitochondrial and un-glutathionylated PDIA1-FLAG masks of enlarged boxed areas from A and D. Single planes with maximal signal intensities in both channels (FLAG and mitoSox) are shown on the left, 3D reconstitution of the whole area on the right. ( C, and F ) Overlay of mitochondrial and glutathionylated PDIA1-FLAG masks of enlarged boxed areas from A and D. Same single planes as in B and E with maximal signal intensities in both channels are shown on the left, 3D reconstitution of the whole area on the right. ( G ) Quantification of un-glutathionylated and glutathionylated PDIA1-FLAG. ( H ) Quantification of associated with mitochondria un-glutathionylated and glutathionylated PDIA1-FLAG. ( I ) Western blot of transfected with PDI FLFL−FLAG cells. PDIA1 is visualized by anti-PDI antibodies. Upper band corresponds to PDI FLFL−FLAG , lower band to endogenous PDIA1. Ratio of PDI FLFL−FLAG /PDIA1 endogenous calculated via densitometry using ImageLab 6.1 (Biorad) Volume Tool with rectangular selection.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Variant Assay, Transfection, FLAG-tag, Expressing, Western Blot, Selection

    S-glutathionylated PDIA1 relocation to ERMCS decreases mitochondrial membrane potential and induces mitochondrial permeability transition pore opening. (A) MLE12 cells were transfected with pcDNA3.1 PDI WT , pcDNA3.1 PDI FLFL , or empty vector (EV). After 24 h, cells were loaded with 1 μM TMRM for 30 min, treated with 20 % CSE for 15 min, and fluorescence intensity (FI) was measured using a CytoFLEX S flow cytometer. Data were analyzed with CytExpert 2.1 software. (B) Bar graphs represent FI. (C, D) Changes in mitochondrial membrane potential were also assessed in stably transfected MCF7 cells expressing PDIWT or PDIFLFL. (C) MCF7 cells were loaded with 1 μM TMRM for 30 min, treated with 20 % CSE, and imaged using a Zeiss LSM880 confocal microscope. Scale bar included. (D) FI was quantified using ImageJ from four different fields, represented as relative intensity to control. (E) Mitochondrial permeability transition pore (MPTP) opening was assessed by monitoring optical density changes in isolated mitochondria after Ca 2+ treatment, with or without cyclosporin A (CsA), using a spectrophotometer at 520 nm. (F) Absorbance changes in CSE-treated PDI WT mitochondria with or without CsA. (G) Absorbance changes in CSE-treated PDIA1 FLFL mitochondria with or without CsA.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: S-glutathionylated PDIA1 relocation to ERMCS decreases mitochondrial membrane potential and induces mitochondrial permeability transition pore opening. (A) MLE12 cells were transfected with pcDNA3.1 PDI WT , pcDNA3.1 PDI FLFL , or empty vector (EV). After 24 h, cells were loaded with 1 μM TMRM for 30 min, treated with 20 % CSE for 15 min, and fluorescence intensity (FI) was measured using a CytoFLEX S flow cytometer. Data were analyzed with CytExpert 2.1 software. (B) Bar graphs represent FI. (C, D) Changes in mitochondrial membrane potential were also assessed in stably transfected MCF7 cells expressing PDIWT or PDIFLFL. (C) MCF7 cells were loaded with 1 μM TMRM for 30 min, treated with 20 % CSE, and imaged using a Zeiss LSM880 confocal microscope. Scale bar included. (D) FI was quantified using ImageJ from four different fields, represented as relative intensity to control. (E) Mitochondrial permeability transition pore (MPTP) opening was assessed by monitoring optical density changes in isolated mitochondria after Ca 2+ treatment, with or without cyclosporin A (CsA), using a spectrophotometer at 520 nm. (F) Absorbance changes in CSE-treated PDI WT mitochondria with or without CsA. (G) Absorbance changes in CSE-treated PDIA1 FLFL mitochondria with or without CsA.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Membrane, Permeability, Transfection, Plasmid Preparation, Fluorescence, Flow Cytometry, Software, Stable Transfection, Expressing, Microscopy, Control, Isolation, Spectrophotometry

    S-glutathionylated PDIA1 causes Cyt c release, apoptosis, and cytotoxicity following CSE exposure . (A – C) Mitochondria were incubated with CSE (20 %)-treated PDIA1 WT or PDIA1 FLFL , with or without CsA and Ca 2+ (150 μM), in the presence of cytosol for 30 min at 37 °C. (A) Mitochondria were pelleted, and the presence of Cyt c in the supernatant was evaluated by Western blotting. PDH antibodies were used to assess mitochondrial input as a loading control. (B) Quantification of Cyt c release relative to control (mean ± SD, ∗∗∗ p < 0.001). (C) Cyt c release after incubation with S-glutathionylated PDIA1 induced caspase activation, measured via DEVD-AFC cleavage after 20 min. (D – F) Caspases are not required for S-glutathionylated PDIA1-induced Cyt c release. Mitochondria were incubated with CSE (20 %)-treated PDIA1 WT or PDIA1 FLFL , with or without zVAD-fmk (10 μM) and Ca 2+ (150 μM), in the presence of cytosol for 30 min at 37 °C. (D) Cyt c levels in the cytosol were analyzed by Western blot. PDH antibodies were used to assess mitochondrial input as a loading control. (E) Relative Cyt c release (mean ± SD, ∗∗∗ p < 0.001). (F) Caspase activity measured via DEVD-AFC cleavage (∗∗∗ p < 0.001). (G) S-glutathionylated PDIA1 enhances CSE-induced cytotoxicity, assessed by MTT assay in MLE12 cells transfected with EV, PDIA1 WT , or PDIA1 FLFL . (H) MTT assay measuring CSE cytotoxicity with or without the GSTP1 inhibitor TLK199.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: S-glutathionylated PDIA1 causes Cyt c release, apoptosis, and cytotoxicity following CSE exposure . (A – C) Mitochondria were incubated with CSE (20 %)-treated PDIA1 WT or PDIA1 FLFL , with or without CsA and Ca 2+ (150 μM), in the presence of cytosol for 30 min at 37 °C. (A) Mitochondria were pelleted, and the presence of Cyt c in the supernatant was evaluated by Western blotting. PDH antibodies were used to assess mitochondrial input as a loading control. (B) Quantification of Cyt c release relative to control (mean ± SD, ∗∗∗ p < 0.001). (C) Cyt c release after incubation with S-glutathionylated PDIA1 induced caspase activation, measured via DEVD-AFC cleavage after 20 min. (D – F) Caspases are not required for S-glutathionylated PDIA1-induced Cyt c release. Mitochondria were incubated with CSE (20 %)-treated PDIA1 WT or PDIA1 FLFL , with or without zVAD-fmk (10 μM) and Ca 2+ (150 μM), in the presence of cytosol for 30 min at 37 °C. (D) Cyt c levels in the cytosol were analyzed by Western blot. PDH antibodies were used to assess mitochondrial input as a loading control. (E) Relative Cyt c release (mean ± SD, ∗∗∗ p < 0.001). (F) Caspase activity measured via DEVD-AFC cleavage (∗∗∗ p < 0.001). (G) S-glutathionylated PDIA1 enhances CSE-induced cytotoxicity, assessed by MTT assay in MLE12 cells transfected with EV, PDIA1 WT , or PDIA1 FLFL . (H) MTT assay measuring CSE cytotoxicity with or without the GSTP1 inhibitor TLK199.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques: Incubation, Western Blot, Control, Activation Assay, Activity Assay, MTT Assay, Transfection

    A schematic illustration of the present report. We identify GSTP1 as a stress-dependent regulator of ER–mitochondria contact site (ERMCS) composition. GSTP1 modulates the S-glutathionylation of PDIA1, which in turn dictates the intra-ER localization of distinct PDIA1 pools, ultimately influencing intraorganellar communication.

    Journal: Redox Biology

    Article Title: Redox signaling-mediated S-glutathionylation of protein disulfide isomerase A1 initiates intrinsic apoptosis and contributes to accelerated aging

    doi: 10.1016/j.redox.2025.103680

    Figure Lengend Snippet: A schematic illustration of the present report. We identify GSTP1 as a stress-dependent regulator of ER–mitochondria contact site (ERMCS) composition. GSTP1 modulates the S-glutathionylation of PDIA1, which in turn dictates the intra-ER localization of distinct PDIA1 pools, ultimately influencing intraorganellar communication.

    Article Snippet: Visually, PDIA1 was more abundant in AEC2s than in AEC1s ( A, third panel, yellow arrow), this observation was confirmed by quantification of PDIA1 signal intensity ( B) and supported by previously reported higher PDIA1 (P4HB) mRNA levels in AEC2s (Human Protein Atlas).

    Techniques:

    Figure 3. Natural source phenethyl isothio- cyanate improves AAT-Z monomer secre- tion and NE inhibitory activity (A) The chemical structure of phenethyl isothio- cyanate (PEITC). (B) Immunoblotting analysis of extracellular AAT-Z in the absence or presence of PEITC in lung cells. IB3null-Z cells were treated with PEITC (5 mM) for 24 h; the cell lysate and culture medium were collected for SDS-PAGE and immunoblotting to measure AAT-Z total secretion. AAT-WT expres- sion was used as control. (C and D) AAT-Z monomer secretion (C) and NE inhibitory activity (D) in the absence or presence of PEITC (5 mM) for 24 h in IB3null-Z cells were measured by using an ELISA assay and NE inhibitory fluorogenic assay, respectively. (E and F) AAT-Z monomer secretion (E) and NE inhibitory activity (F) in the absence or presence of PEITC from Huh7.5null-Z were measured by ELISA and NE inhibitory fluorogenic assays, respectively. (G and H) The impact of PEITC in AAT-Z HLCs. Cells were treated by PEITC with 5, 10, or 20 mM for 24 h. The AAT-Z secreted monomer (G) and NE inhibitory activity (H) in the absence or presence of PEITC were measured by ELSIA assay and NE inhibitory fluorogenic assay. (I and J) A PDI general inhibitor eliminates PEITC correction of AAT-Z. IB3null-Z cells expressing AAT-Z were treated with the PDI inhibitor 16F16 (20 mM) in the absence or presence of PEITC (5 mM) for 24 h. Cell culture medium was collected, and AAT-Z secreted monomer (I) and NE inhibitory activity measured (J). (K and L) PDIA4 silencing eliminates PEITC correction of AAT-Z secretion and NE inhibitory activity. PDIA1, PDIA3, or PDIA4 expression were silenced by siRNA transfection in IB3null-Z

    Journal: Cell reports. Medicine

    Article Title: Spatial covariance reveals isothiocyanate natural products adjust redox stress to restore function in alpha-1-antitrypsin deficiency.

    doi: 10.1016/j.xcrm.2024.101917

    Figure Lengend Snippet: Figure 3. Natural source phenethyl isothio- cyanate improves AAT-Z monomer secre- tion and NE inhibitory activity (A) The chemical structure of phenethyl isothio- cyanate (PEITC). (B) Immunoblotting analysis of extracellular AAT-Z in the absence or presence of PEITC in lung cells. IB3null-Z cells were treated with PEITC (5 mM) for 24 h; the cell lysate and culture medium were collected for SDS-PAGE and immunoblotting to measure AAT-Z total secretion. AAT-WT expres- sion was used as control. (C and D) AAT-Z monomer secretion (C) and NE inhibitory activity (D) in the absence or presence of PEITC (5 mM) for 24 h in IB3null-Z cells were measured by using an ELISA assay and NE inhibitory fluorogenic assay, respectively. (E and F) AAT-Z monomer secretion (E) and NE inhibitory activity (F) in the absence or presence of PEITC from Huh7.5null-Z were measured by ELISA and NE inhibitory fluorogenic assays, respectively. (G and H) The impact of PEITC in AAT-Z HLCs. Cells were treated by PEITC with 5, 10, or 20 mM for 24 h. The AAT-Z secreted monomer (G) and NE inhibitory activity (H) in the absence or presence of PEITC were measured by ELSIA assay and NE inhibitory fluorogenic assay. (I and J) A PDI general inhibitor eliminates PEITC correction of AAT-Z. IB3null-Z cells expressing AAT-Z were treated with the PDI inhibitor 16F16 (20 mM) in the absence or presence of PEITC (5 mM) for 24 h. Cell culture medium was collected, and AAT-Z secreted monomer (I) and NE inhibitory activity measured (J). (K and L) PDIA4 silencing eliminates PEITC correction of AAT-Z secretion and NE inhibitory activity. PDIA1, PDIA3, or PDIA4 expression were silenced by siRNA transfection in IB3null-Z

    Article Snippet: Inc Cat # GA1T-80A mouse anti-human AAT monoclonal antibody 16F8 This paper Scripps Research Antibody Development and Production Core mouse anti-human AAT monoclonal antibody 2C1 Hycult Biotech Cat # HM2289 mouse anti-human AAT monoclonal antibody QED Bioscience Inc. Cat # 13701 PDIA1 antibody Cell Signaling Technology Cat # 2466, RRID: AB_2298935 PDIA3 antibody Sigma-Aldrich Cat # 1410582 PDIA4 antibody Abcam Cat # ab155800 GAPDH antibody Abcam Cat # ab8245 Goat anti-Mouse HRP antibody Thermo Fisher Scientific Cat # 32230, RRID:AB_1965958 Goat anti-Rabbit HRP antibody Thermo Fisher Scientific Cat# 32260, RRID:AB_1965959 Mouse anti-Goat HRP antibody Thermo Fisher Scientific Cat# 31400, RRID:AB_228370 Human neutrophil elastase Innovative Research Cat # IHUELASD100UG Neutrophil elastase fluorescence substrate 2Rh110 Cayman Chemical Cat # 11675 polystyrene high binding plate Corning Cat # 3690 Protein A beads Thermo Fisher Scientific Cat # 20334 Streptavidin beads Thermo Fisher Scientific Cat # 20349 Protein G beads Thermo Fisher Scientific Cat # 20397 FuGENE6 transfection reagent Promega Cat #E2691 Lipofectamine RNAiMAX Thermo Fisher Scientific Cat # 13778150 PDIA1 siRNA Thermo Fisher Scientific Cat # 110789 PDIA3 siRNA Thermo Fisher Scientific Cat # 107677 PDIA4 siRNA Thermo Fisher Scientific Cat # 107580 DMEM medium Corning Cat # 15-013-CM F12 medium Sigma-Aldrich Cat #N6658-500ML LHC-8 medium Thermo Fisher Scientific Cat # 12679-015 Fetal bovine serum Thermo Fisher Scientific Cat # SH30396.03 penicillin streptomycin (P/S) Thermo Fisher Scientific Cat # 15140-122 (Continued on next page) Cell Reports Medicine 6, 101917, January 21, 2025 e1

    Techniques: Activity Assay, Western Blot, SDS Page, Control, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Transfection