Journal: Biomolecules

Article Title: High Glucose in Diabetic Hyperglycemia Perturbs Lymphocyte SERCA-Regulated Ca 2+ Stores with Accompanying ER Stress and Signaling Dysfunction

doi: 10.3390/biom15070987

Figure Lengend Snippet: High glucose exposure induces SERCA pumps and core ER stress mediators in Jurkat lymphocytes that precede detectable changes in agonist-induced ER Ca 2+ release. ( A ) Representative Western blot showing GRP78 expression levels in Jurkat lymphocytes exposed to normal (5.5 mM), intermediate high (15 mM) and high (25 mM) glucose levels for 7 days. Also shown is gel densitometry plot depicting changes in GRP78 levels normalized to β-actin expression levels in normal (5.5 mM, black bar), intermediate high (15 mM, green bar) and high (25 mM, pink bar) glucose levels. ( B ) RT-qPCR experiments performed on Jurkat lymphocytes incubated in high glucose (25 mM) for 3 days depicting relative mRNA levels for SERCA 2, SERCA 3, GRP78, XBP1, CHOP and PDIA4. Also shown are control experiments of cells grown in normal (5.5 mM) glucose levels and mannitol (25 mM). All mRNA levels were normalized to control GAPDH mRNA. One-way ANOVA followed by Tukey’s and Dunnett’s multiple comparison tests were used to analyze Western blot data for GRP78 expression levels and RT-qPCR data with n = 3, respectively. Asterisks denote statistical significance with * p < 0.05, ** p < 0.005, *** p < 0.001 and ns is not significant p > 0.05.

Article Snippet: Real-time quantitative PCR was performed using TaqManTM Fast Advanced Master Mix and TaqMan ® Assay primer/probe (fluorogenic probe-primer combinations specific for the target gene; Assay ID: Hs00544877_m1 ATP2A2, Hs01024563_m1 ATP2A3, Hs02758991_g1 GAPDH, Hs00231936_m1 XBP1, Hs00358796_g1 DDIT3/CHOP, Hs00607129_gH HSPA5/GRP78, Hs01115905_m1 PDIA4/ERP72) (Thermo Fisher Scientific).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Incubation, Control, Comparison

Journal: Nutrition & Diabetes

Article Title: ER stress in rodent islets of Langerhans is concomitant with obesity and β-cell compensation but not with β-cell dysfunction and diabetes

doi: 10.1038/nutd.2013.35

Figure Lengend Snippet: List of genes on the Taqman low-density array (TLDA) whose mRNA expression was determined by qPCR

Article Snippet: Protein disulphide isomerase family A, member 4 , Pdia4 , ERp72/ERP70 , NM053849.1 , Rn00587766_m1.

Techniques: TLDA Assay, Expressing, Binding Assay

Journal: Cell Death & Disease

Article Title: PDIA4 confers resistance to ferroptosis via induction of ATF4/SLC7A11 in renal cell carcinoma

doi: 10.1038/s41419-023-05719-x

Figure Lengend Snippet: HK-2 and RCC cell lines ACHN, 786-O, and OSRC2 were treated with DMSO or Sal (2 µM), and lysates were subjected to thermolysin digestion. A Coomassie blue staining. B Enrichment of proteins in the protected band (red frame in ( A )) revealed by mass spectrometry analysis. C Western blot assay of cell lysates using antibodies against PDIA4. 786-O and ACHN cells were treated with DMSO or Sal (2 µM). D Western blot assay of cell lysates using antibodies against PDIA4. The volume of β-actin was used as the loading control. 786-O cells were treated with DMSO or Sal (2 µM) and subjected to protein synthesis inhibitor CHX as indicated. E Western blotting assay of cell lysates using antibodies against PDIA4 (left). The curve of protein degradation rate (right). Data are present as mean ± S.E.M., n = 5. P values were calculated using Student’s t -test. *** P < 0.001. 786-O cells were treated with DMSO or Sal (2 µM) in the presence of proteasome inhibitor MG-132 or autophagy inhibitor Baf-A1 or CQ. F Western blot assay of cell lysates using antibodies against PDIA4. 786-O cells were treated with DMSO or Sal (2 µM). G Immunofluorescence staining using antibodies against LC3 (green) and PDIA4 (red). Scale bar: 10 μm. Stable ATG5-silencing down or scramble control 786-O cells were treated with DMSO or Sal (2 µM). H Western blot assay of cell lysates using antibodies against ATG5, SQSTM1, PDIA4, or LC3. I Co-immunoprecipitation assay of cell lysates in Sal-treated 786-O cells. IP was performed using antibodies against PDIA4 and immunoblotting with antibodies against SQSTM1.

Article Snippet: PCR products were then ligated into a pLV-EF1α-MCS-IRES-Bsd vector (Biosettia) separately. shRNAs against PDIA4 or ATG5 were constructed using pLKO.1-TRC cloning vector (Addgene) according to the manufacturer’s protocol.

Techniques: Staining, Mass Spectrometry, Western Blot, Control, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: PDIA4 confers resistance to ferroptosis via induction of ATF4/SLC7A11 in renal cell carcinoma

doi: 10.1038/s41419-023-05719-x

Figure Lengend Snippet: Stable PDIA4-silencing-down or scramble control 786-O cells were treated with Sal (2 µM). A Western blot assay of cell lysates using antibodies against PDIA4 and GPX4. B Flow cytometry analysis showing lipid ROS by means of fluorogenic reaction with BODIPY 589/591 C11 in stable PDIA4-silencing-down or scramble control 786-O cells. Stable PDIA4-silencing-down or scramble control 786-O cells were treated with Sal (2 µM) and transfected with GPX4-overexpressing or control plasmid. C Cell viability assay and D level of lipid peroxidation measured by MDA assay. Stable PDIA4- or scramble control-silencing down 786-O cells were treated with Sal (2 µM) or separate ferroptosis inducers, RSL3 (1 µM) and erastin (2 µM) for 24 h. E Cell viability assay and F level of lipid peroxidation measured by MDA assay. Stable PDIA4- or control MCS-overexpressing 786-O cells were treated with Sal (2 µM) or separate ferroptosis inducers, RSL3 (1 µM) and erastin (2 µM) for 48 h. G Western blot assay of cell lysates using antibodies against PDIA4 and GPX4. H Flow cytometry analysis showing lipid ROS by means of fluorogenic reaction with BODIPY C11. I Cell viability assay and J level of lipid peroxidation measured by MDA assay. Data are present as mean ± S.E.M., n = 5 independent repeats. Student’s t -test was performed in ( B ) and ( H ). One-way ANOVA analysis was performed in ( C ), ( D ), ( E ), ( F ), ( I ), and ( J ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: PCR products were then ligated into a pLV-EF1α-MCS-IRES-Bsd vector (Biosettia) separately. shRNAs against PDIA4 or ATG5 were constructed using pLKO.1-TRC cloning vector (Addgene) according to the manufacturer’s protocol.

Techniques: Control, Western Blot, Flow Cytometry, Transfection, Plasmid Preparation, Viability Assay, Multiple Displacement Amplification

Journal: Cell Death & Disease

Article Title: PDIA4 confers resistance to ferroptosis via induction of ATF4/SLC7A11 in renal cell carcinoma

doi: 10.1038/s41419-023-05719-x

Figure Lengend Snippet: RNA-seq was performed in stable PDIA4-silencing-down 786-O cells, with scramble control 786-O cells as control. A Scatter plot of differential expression genes. B Overlay of downregulated genes (shPDIA4 vs scramble control) with known regulatory genes in ferroptosis. C Real-time PCR and western blot assay for SLC7A11 expression in stable PDIA4-silencing-down or scramble control 786-O cells. Stable PDIA4-silencing-down or scramble control 786-O cells were treated with Sal (2 µM). D Western blot assay of cell lysates using antibodies against p-PERK/PERK and ATF4. Stable PDIA4- or control MCS-overexpressing 786-O cells were treated with Sal (2 µM) for 24 h. E , F Western blot assay of cell lysates using antibodies against SLC7A11, PDIA4, phosphorylated PERK, and ATF4. 786-O cells were transfected with ATF4 overexpressing plasmid or control plasmid in the presence with or without Sal (2 µM). G Cell viability assay and H level of lipid peroxidation measured by MDA assay. I Western blot assay of PDIA4, PERK, ATF4, and SLC7A11 in cancerous and adjacent normal tissue resected from patients of RCC, and the statistical analyses. Data are present as mean ± S.E.M., n = 5. Student’s t -test was performed in ( C ) and ( I ), and one-way ANOVA analysis was performed in ( G ) and ( H ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: PCR products were then ligated into a pLV-EF1α-MCS-IRES-Bsd vector (Biosettia) separately. shRNAs against PDIA4 or ATG5 were constructed using pLKO.1-TRC cloning vector (Addgene) according to the manufacturer’s protocol.

Techniques: RNA Sequencing, Control, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation, Viability Assay, Multiple Displacement Amplification

Journal: Cell Death & Disease

Article Title: PDIA4 confers resistance to ferroptosis via induction of ATF4/SLC7A11 in renal cell carcinoma

doi: 10.1038/s41419-023-05719-x

Figure Lengend Snippet: 2 × 10 6 786-O cells were administrated subcutaneously to BALB/c nude mice to establish xenograft mouse models. Animals were randomly divided into Sal treatment and vehicle control groups ( n = 8), with the administration of Sal (5 mg/kg BW, intraperitoneally, every other day) or solvent vehicle from the day when the size of the tumor reaches 50 mm 3 . A Schematic diagram of in vivo experimental design. B Tumor growth curve. C Representative images of isolated tumor (left) and the result of statistical analysis on the size data (right). D Level of lipid peroxidation measured by MDA assay in the tumor homogenates. E Representative images (up) and statistic data (bottom) of 4-HNE assay and F Representative images (up) and statistic data (bottom) of Prussian blue staining. G Representative images of H&E, IHC staining of Ki67, and cleaved caspase-3 staining (up) and statistic data (bottom). Scale bar: 50 µm. H Western blot analysis on the tumor tissue homogenates by using antibodies against PDIA4, ATF4, SLC7A11, and GPX4. The right panel is the result of the statistical analysis of the integral optical density of separate bands. Data are present as mean ± S.E.M., n = 5. P values were calculated using a two-tailed unpaired Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: PCR products were then ligated into a pLV-EF1α-MCS-IRES-Bsd vector (Biosettia) separately. shRNAs against PDIA4 or ATG5 were constructed using pLKO.1-TRC cloning vector (Addgene) according to the manufacturer’s protocol.

Techniques: Control, Solvent, In Vivo, Isolation, Multiple Displacement Amplification, Staining, Immunohistochemistry, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PDIA4 confers resistance to ferroptosis via induction of ATF4/SLC7A11 in renal cell carcinoma

doi: 10.1038/s41419-023-05719-x

Figure Lengend Snippet: A Comparison of PDIA4 gene expression between normal kidney tissue and a cancerous sample of ccRCCs. Data were obtained from Gumz, Beroukhim, and Yusenko database sets, respectively. B Comparison of the gene expression data of PDIA4, ATF4, SLC7A11, and PERK between normal and carcinoma tissues obtained from the TCGA database. C , D Correlation assay on the gene expression data of PDIA4 and PERK or PDIA4 and SLC7A11 in RCC patients obtained from TCGA database. E Kaplan–Meier survival plot of PDIA4 high expression and low/medium expression groups. Data were obtained from the TCGA database.

Article Snippet: PCR products were then ligated into a pLV-EF1α-MCS-IRES-Bsd vector (Biosettia) separately. shRNAs against PDIA4 or ATG5 were constructed using pLKO.1-TRC cloning vector (Addgene) according to the manufacturer’s protocol.

Techniques: Comparison, Gene Expression, Two-Photon Excitation Fluorescence Cross-Correlation Assay, Expressing

Journal: Cell Death & Disease

Article Title: PDIA4 confers resistance to ferroptosis via induction of ATF4/SLC7A11 in renal cell carcinoma

doi: 10.1038/s41419-023-05719-x

Figure Lengend Snippet: Sal exhibits pro-ferroptic effect in RCC via downregulating PDIA4. Administration of Sal stimulates autophagic degradation and thus inhibits PDIA4. Downregulation of PDIA4 suppresses ATF4 and SLC7A11, thereby inactivates GPX4 and promotes ferroptosis.

Article Snippet: PCR products were then ligated into a pLV-EF1α-MCS-IRES-Bsd vector (Biosettia) separately. shRNAs against PDIA4 or ATG5 were constructed using pLKO.1-TRC cloning vector (Addgene) according to the manufacturer’s protocol.

Techniques:

Journal: eLife

Article Title: Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

doi: 10.7554/eLife.37168

Figure Lengend Snippet: Protein targets that are covalently modified by 147 . 10.7554/eLife.37168.009 Table 1—source data 1. Excel spreadsheet showing the competition ratio data for all 90 proteins that were identified as targets of 147 in every replicate and every cell type. 10.7554/eLife.37168.010 Table 1—source data 2. Excel spreadsheet showing all the proteins identified among the affinity-purified targets of 147 .

Article Snippet: Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Techniques: Modification, Affinity Purification

Journal: eLife

Article Title: Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

doi: 10.7554/eLife.37168

Figure Lengend Snippet: ( A ) Proportion of ER-localized proteins covalently labeled by 147 or fragment electrophiles reported in . The ER-localized proportion for each electrophile indicated in red. The numbers in parentheses indicate the number of ER-localized proteins in a given electrophile’s target list on the left and the total number of proteins in the target list on the right. The electrophiles from are denoted with a B- followed by the compound numbers used in that work. ( B ) Bar graph showing activation of the ERSE.FLuc reporter in HEK293T cells treated with 147 (10 µM), thapsigargin (Tg; 0.5 µM) or the indicated dose of acetaminophen for 18 hr. Error bars show SEM for 3 independent experiments. ( C ) Immunoblot of PDI4 in 147–20 affinity purified proteins from HEK293T cells treated with 147 (10 µM), or 147–20 (10 µM), or the combination of 147–20 (10 µM) and 147 (50 µM), or the combination of 147–20 (10 µM) and 147–4 (50 µM) for 18 hr. The relative recovery of PDIA4 under these different conditions is indicated below the immunoblot.

Article Snippet: Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Techniques: Labeling, Activation Assay, Western Blot, Affinity Purification

Journal: eLife

Article Title: Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

doi: 10.7554/eLife.37168

Figure Lengend Snippet: ( A ) Mechanistic model of 147 -dependent ATF6 activation, as described in the main text. The red component of the figure shows how pharmacologic modulation of ATF6 by 147 or CP7 influences ATF6 activity. ( B ) Immunoblot showing lysates prepared from HeLa cells stably expressing FLAG-tagged ATF6 treated for 6 hr with 147 (10 µM) or tunicamycin (Tm; 1 mg/mL). Lysates were separated by non-reducing or reducing SDS-PAGE prior to immunoblotting. The bands representing oxidized and reduced ATF6 are indicated. Note the increased ATF6 migration observed in reducing gels for Tm-treated cells, reflecting the inhibited N-linked glycosylation of ATF6 in these samples. ( C ) Graph showing relative activation of the ERSE.Fluc ATF6 reporter in HEK293T cells co-treated for 18 hr with 147 (10 µM) and increasing concentrations of CP7 , as indicated. Error bars show SEM for three technical replicates. ( D ) Bar graph showing BiP expression in HEK293T cells stably expressing non-silencing, PDIA1 , PDIA3, PDIA4 , PDIA5 , or PDIA6 shRNA, as indicated. BiP expression in the PDI-depleted cells is shown relative to cells expressing non-silencing shRNA. Error bars show SEM for 3 independent experiments. *indicates p<0.05. ( E ) Bar graph showing BiP expression in HEK293T cells expressing non-silencing, PDIA1 , PDIA3, PDIA4 , PDIA5 , or PDIA6 shRNA treated for 6 hr with or without 147 (10 µM). BiP expression levels for samples treated with 147 are normalized to the corresponding vehicle-treated controls. Un-normalized data are shown in . Error bars show SEM for 3 independent experiments. *indicates p<0.05. ( F ) Bar graph showing BiP expression in HEK293T cells expressing non-silencing, PDIA1 , PDIA3, PDIA4 , PDIA5 , or PDIA6 shRNA treated for 6 hr with or without thapsigargin (Tg; 0.5 µM). BiP expression levels for samples treated with Tg are normalized to the corresponding vehicle-treated controls. Un-normalized data are shown in Error bars show SEM for 3 independent experiments.

Article Snippet: Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Techniques: Activation Assay, Activity Assay, Western Blot, Stable Transfection, Expressing, SDS Page, Migration, Glycoproteomics, shRNA

Journal: eLife

Article Title: Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

doi: 10.7554/eLife.37168

Figure Lengend Snippet: ( A ) Bar graph showing expression of PDIA1 , PDIA3 , PDIA4 , PDIA5 , or PDIA6 in HEK293T cells depleted for the same PDI. The dashed line reflects expression of each PDI in control cell lines expressing non-silencing shRNA. Error bars show ± 95% confidence interval. ( B ), ( C ) Un-normalized data for and , respectively. Error bars show SEM for 3 independent experiments.

Article Snippet: Proteins were transferred from gel slabs to nitrocellulose and blotted using mouse anti-FLAG M2 antibody (Sigma) or rabbit anti-PDIA4 antibody (Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Techniques: Expressing, Control, shRNA