Journal: Physiological Reports

Article Title: Mechanism of paraventricular nucleus H 2 S on PERK / TXNIP / NLRP3 pathway in male spontaneously hypertensive rats

doi: 10.14814/phy2.70861

Figure Lengend Snippet: Effects of SAMe and GSK on the expression of CBS, PERK/TXNIP/NLRP3 pathway‐related proteins, and inflammatory cytokines in the PVN of SHR. (a) Protein electropherograms of CBS, p‐PERK/PERK, TXNIP, NLRP3, ASC, pro‐Caspase‐1, Caspase‐1 p20, IL‐1β, TNF‐α, and IL‐18. (b–k) Results of statistical analysis of the related proteins and inflammatory factors in the PVN of each group of rats, respectively. All data are expressed as a mean ± SD. n = 6 in each group. SAMe (an endogenous H 2 S agonist), GSK (a PERK inhibitor).

Article Snippet: The membrane was sealed with BSA or skimmed milk powder for 1 h. Following the sealing, the membrane was exposed to the following primary antibodies at 4°C in the refrigerator overnight: anti‐β‐actin (1:1800; TA‐09; ZSGB‐BIO), CBS (1:1000; sc‐515180; Santa Cruz Biotechnology), PERK (1:2000; WL03378; WanLei), p‐PERK (1:2000; bs‐3330R; Bioss), TXNIP (1:1500; ab232330; Abcam), NLRP3 (1:2000; ab314905; Abcam), ASC (1:1000; AF6234; Beyotime Biotechnology ), pro‐Caspase‐1 (1:1500; sc‐56036; Santa Cruz Biotechnology), Caspase‐1 p20 (1:2000; WL02996a; WanLei), IL‐1β (1:1000; ab234437; Abcam), TNF‐α (1:1000; ab215188; Abcam), and IL‐18 (1:2000; AF5207; Beyotime Biotechnology ).

Techniques: Expressing

Journal: Physiological Reports

Article Title: Mechanism of paraventricular nucleus H 2 S on PERK / TXNIP / NLRP3 pathway in male spontaneously hypertensive rats

doi: 10.14814/phy2.70861

Figure Lengend Snippet: Effect of SAMe and GSK treatment on p‐PERK expression in the PVN of SHR. (a) Immunofluorescence staining of p‐PERK in the rat PVN, scale bar = 50 μm. (b) Statistical graph in average absorbance of p‐PERK in the rat PVN. All data are expressed as a mean ± SD. n = 4 in each group. SAMe (an endogenous H 2 S agonist), GSK (a PERK inhibitor).

Article Snippet: The membrane was sealed with BSA or skimmed milk powder for 1 h. Following the sealing, the membrane was exposed to the following primary antibodies at 4°C in the refrigerator overnight: anti‐β‐actin (1:1800; TA‐09; ZSGB‐BIO), CBS (1:1000; sc‐515180; Santa Cruz Biotechnology), PERK (1:2000; WL03378; WanLei), p‐PERK (1:2000; bs‐3330R; Bioss), TXNIP (1:1500; ab232330; Abcam), NLRP3 (1:2000; ab314905; Abcam), ASC (1:1000; AF6234; Beyotime Biotechnology ), pro‐Caspase‐1 (1:1500; sc‐56036; Santa Cruz Biotechnology), Caspase‐1 p20 (1:2000; WL02996a; WanLei), IL‐1β (1:1000; ab234437; Abcam), TNF‐α (1:1000; ab215188; Abcam), and IL‐18 (1:2000; AF5207; Beyotime Biotechnology ).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Cancers

Article Title: Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer.

doi: 10.3390/cancers14122949

Figure Lengend Snippet: Figure 6. Mifepristone activates the HRI kinase pathway. (A): Mouse embryonic fibroblast generated from PERK knockout mice (PERK-KO-DR) and mouse embryonic fibroblast generated from GCN2 knockout mice (GCN2-KO-DR) were incubated with 20 µM of mifepristone for 6 and 12 h. Vehicle (V) and TN (2 µM-Tunicamycin) treatments were performed for 12 h and lysates were probed with indicated antibodies. β-actin was used as loading control. (B): RMG1 cells were transiently transfected with scrambled (Scr) and small interfering RNAs (siRNAs) targeting HRI (si-HRI) and PKR (si-PKR) for 48 h followed by treatment of 20 µM of mifepristone for 6 and 10 h. Protein lysates were then subjected to immunoblotting with indicated antibodies. β-actin was used as loading control. All experiments were performed in at least two biological replicates. Uncropped immunoblots can be found in Figure S12.

Article Snippet: The primary antibodies, purchased from Cell Signaling Technology (Danvers, MA, USA), included PARP (#9542), total caspase-3 (#9665), cleaved caspase-3 (#9661), Grp78 (#3177), CHOP (#5554), β-actin (#3700), ubiquitin (#3933), ATF6 (#65880), PERK (#3192), ATF4 (#11815), IRE1α (#3294), XBP1 (#12782), GCN2 (#3302) and PKR (#12297), except p-eIF2α (ab32157, Abcam) and p-IRE1α (nb100-2323, Novus, Centennial, CO, USA).

Techniques: Generated, Knock-Out, Incubation, Control, Transfection, Western Blot

Journal: Cancers

Article Title: Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer.

doi: 10.3390/cancers14122949

Figure Lengend Snippet: Figure 8. Putative mechanism for synergistic cytotoxicity between CB-5083 and mifepristone. Treat- ment with VCP inhibitor-CB-5083 activates the activating transcription factor 6 (ATF6) branch and the protein kinase R-like endoplasmic reticulum kinase (PERK) branch. Activation of ATF6 can result in the induction of glucose-regulated protein 78 (Grp78). Induction of Grp78 is considered a pro-survival response. Similarly, PERK activation can ultimately induce CCAAT/enhancer-binding protein homologous protein (CHOP). Enhanced expression of CHOP can be a pro-apoptotic response. Mifepristone treatment inhibits the ATF6 branch and enhances CHOP expression through the acti- vation of the heme-regulated inhibitor (HRI) kinase pathway. The combination of these two effects could result in the enhanced cytotoxicity seen with CB-5083 and mifepristone combination.

Article Snippet: The primary antibodies, purchased from Cell Signaling Technology (Danvers, MA, USA), included PARP (#9542), total caspase-3 (#9665), cleaved caspase-3 (#9661), Grp78 (#3177), CHOP (#5554), β-actin (#3700), ubiquitin (#3933), ATF6 (#65880), PERK (#3192), ATF4 (#11815), IRE1α (#3294), XBP1 (#12782), GCN2 (#3302) and PKR (#12297), except p-eIF2α (ab32157, Abcam) and p-IRE1α (nb100-2323, Novus, Centennial, CO, USA).

Techniques: Activation Assay, Binding Assay, Expressing

Journal: Oncogene

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

doi: 10.1038/onc.2010.153

Figure Lengend Snippet: (A) PERK protein levels were measured by immunoprecipitation (IP) followed by Western blot analysis in the following cell lines: MCF10A (1), MCF7 (2), T47D (3), MDA-MB231 (4), MDA-MB468 (5), TE3 (6), TE7 (7), KYSE 520 (8). (B) PERK protein levels following shRNA targeting of PERK. (C) Parental MDA-MB468 cell line, shPERK-transduced cells (shPERK), and shPERK-transduced cells reconstituted with mouse Myc-PERK (+mPERK) were treated with 2μg/ml tunicamycin for the indicated intervals. Western analysis for ATF4, CHOP, or β-actin. (D) Volume of orthotopic tumors formed from the mouse mammary tumor-derived cells transduced in vitro with empty vector virus (Neu/PERK loxP/loxP ) or Cre-expressing retrovirus (Neu/PERK Δ/Δ ) 28 days post-transplant (n=4). Representative image of tumors are provided. All p-values determined by Student t-test. (E) Western analysis of transgenic ErbB2 and PERK expression following infection of mouse mammary tumor-derived cells with control (Neu/PERK loxP/loxP ) or Cre-expressing retrovirus (Neu/PERK Δ/Δ ). Thapsigargin treatment (50nM, 1h) was used to demonstrate that PERK is functional.

Article Snippet: Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19 ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Techniques: Immunoprecipitation, Western Blot, shRNA, Derivative Assay, In Vitro, Plasmid Preparation, Virus, Expressing, Transgenic Assay, Infection, Control, Functional Assay

Journal: Oncogene

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

doi: 10.1038/onc.2010.153

Figure Lengend Snippet: (A) MDA-MB468 cells were infected with control shRNA or anti-PERK shRNA for the indicated intervals. Cells were pulsed with BrdU 45 min prior to harvest for FACS analysis. (B) Kinetics of growth of the MDA-MB468 parental cell line, control shRNA-(shControl) or shPERK-transduced cells (shPERK), and shPERK-transduced cells reconstituted with mouse Myc-PERK (+mPERK). PERK protein levels following expression of shRNA targeting human PERK and reconstitution with mouse Myc-PERK are shown. (C) Proliferation rates in mammary gland sections from control (PERK loxP/loxP ) and mammary gland-specific PERK knockout mice (PERK Δ/Δ ) on pregnancy day 16 (P16) and lactation day 3 (L3) were determined by immunohistochemistry for BrdU (animals were injected with BrdU 1 h prior to being euthanized). (D) Quantification of BrdU-positive cells from (C) is shown; error bars indicate S.D. among 3 animals, 5 acini were counted per animal.

Article Snippet: Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19 ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Techniques: Infection, Control, shRNA, Expressing, Knock-Out, Immunohistochemistry, Injection

Journal: Oncogene

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

doi: 10.1038/onc.2010.153

Figure Lengend Snippet: (A) Immunofluorescence staining for DNA damage-induced foci containing phospho-ATM and phospho-Chk2 following acute PERK knockdown (72 h after infection) in MDA-MB468 cells. (B) Quantification of phospho-ATM positive cells (>3 foci) is shown; error bars indicate S.D. from 3 slides, 5 fields were counted per slide. p-value was determined by Student t-test. (C) Western analysis of DNA damage response-associated markers following PERK knockdown. (D) IP/kinase assays assessing CDK2-dependent phosphorylation of histone H1 (bottom panel). CDK2 complexes were immunoprecipitated from MDA-MB 468 cells treated as indicated. PERK levels were assessed by IP/immunoblot and CDK2 recovery in precipitates was assessed by CDK2 immunoblot (middle panel).

Article Snippet: Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19 ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Techniques: Immunofluorescence, Staining, Knockdown, Infection, Western Blot, Phospho-proteomics, Immunoprecipitation

Journal: Oncogene

Article Title: PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

doi: 10.1038/onc.2010.153

Figure Lengend Snippet: (A) Quantitative real time PCR analysis of Nrf2 target genes NQO1 and GCLC in the indicated cell lines asynchronously proliferating under standard conditions. (B) Purified recombinant Nrf2-Neh2 domain of WT, T80A, S40A or T80A/S40A, was incubated with purified recombinant ΔN-PERK in the in vitro kinase assay. Phosphorylated Nrf2-Neh2 was detected by autoradiography (upper panel). (C) 293T cells were transfected with WT Nrf2 or Nrf2-T80A. 24 hours after transfection, cells were left untreated (C) or treated with tunicamycin (Tu) for 2 hours followed by immunoprecipitation with anti-Nrf2 antibody. Threonine phosphorylation was detected using a phospho-Thr reactive antibody. Nrf2 in the IP and the whole cell lysate (WCL) was detected with Nrf2 specific antibody. (D) Proliferation of the indicated cell lines was assessed by a 6-day growth curve under standard tissue culture conditions as described in materials and methods. PERK levels were detected by IP/Western blot analysis. (E) Oxidized guanine in damaged DNA was detected by a FITC-conjugated 8-OxoG binding peptide in PERK knockdown cells infected with pBabe control vector (shPERK) or Nrf2-HA (shPERK Nrf2-HA). Quantification of 8-OxoG positive cells is provided. Error bars represent S.D. from 3 experiments. (F) 8-OxoG was detected in PERK knockdown cells transfected with scramble siRNA (Scrm), or keap1 siRNA (sikeap1). Error bars in graphs represent S.D. from 3 experiments. Western blot panels demonstrate levels of Nrf2-HA and Keap1 in PERK knockdown cells.

Article Snippet: Antibodies used for immunobloting analysis, immunofluorescence and IHC included PERK (Rockland Immunochemicals); human ATF4, histone H3 (tri methyl K9), phospho-Chk2 (Thr68) (Abcam); human CHOP (Affinity Bioreagents), β-actin (Sigma, AC-15), Nrf2, Keap 1, CDK2, and p19 ARF (Santa Cruz Biotechnology); γ-H2AX (Ser139), phospho-eIF2α, eIF4E, Cdc25A, phospho-Tyr15 CDK2, phospho-Thr160 CDK2, phospho-Thr (Cell signaling); troma-1 (Developmental Studies Hybridoma Bank, University of Iowa), ErbB2 (Calbiochem), Chk2 (BD Pharmingen), eIF2α (BioSource), phospho-ATM (Millipore).

Techniques: Real-time Polymerase Chain Reaction, Purification, Recombinant, Incubation, In Vitro, Kinase Assay, Autoradiography, Transfection, Immunoprecipitation, Phospho-proteomics, Western Blot, Binding Assay, Knockdown, Infection, Control, Plasmid Preparation

Journal: Autophagy

Article Title: Direct homophilic interaction of LAMP2A with the two-domain architecture revealed by site-directed photo-crosslinks and steric hindrances in mammalian cells

doi: 10.1080/15548627.2021.1911017

Figure Lengend Snippet: The N-domain truncation of mouse LAMP2A impairs the CMA activity. (A) Domain architectures and orientations of five LAMP family proteins. The core domain common to LAMPs 1–5 has four conserved cysteine residues that can form two disulfide bonds (pink). Both LAMP1 and LAMP2 have two core domains, whereas LAMP3 and LAMP4 have a mucin-like domain (gray) in addition to the core domain [ , ] and LAMP5 is composed of the single core domain . (B) Schematic representations of the full-length and N-domain truncated LAMP2A proteins used in this study. (C, D) rbc1cc1 −/- MEFs either with or without LAMP2 (generated by guide RNA #2) were transfected with GAPDH-HT. The full-length or truncated FLAG-LAMP2A (the N-terminally FLAG-tagged) was expressed together with GAPDH-HT in LAMP2-deficient rbc1cc1 −/- MEFs. The fluorescence images after labeling with TMR-HT ligand (red) for 18–20 h are shown. Nuclei were stained with Hoechst (blue). (D) Quantitative analysis of GAPDH-HT puncta. n = 50, 42, 51, 47. ***P < 0.001. Essentially the same results were obtained in three independent experiments, including one using guide RNA #1. (E) Quantitative analysis of GAPDH-HT puncta upon expression of the full-length or truncated LAMP2A, which is not tagged (left panel, n = 51, 59), or the C-terminally FLAG-tagged LAMP2A (right panel, n = 64, 53), in LAMP2-deficient rbc1cc1 −/- MEFs. (F) GAPDH-HT was transiently expressed in rbc1cc1 −/- MEFs (lane 2) or in the clones of LAMP2-deficient rbc1cc1 −/- MEFs stably expressing the full-length (FL) or truncated (TR) N-terminally FLAG-tagged LAMP2A (lanes 3, 4 and 5, 6, respectively). As a control, rbc1cc1 −/- MEFs were used without transfection of GAPDH-HT (lane 1). Twenty-four hours after the transfection of GPADH-HT, biotin-HT ligand was added to the medium and the cells were cultured for an additional 24 h (lanes 3–6). The proteins in the lysates and retrieved by NeutrAvidin beads (Pulldown) were analyzed by immunoblotting using an anti-HaloTag protein antibody, and reprobed with an anti-tubulin antibody. Closed and open triangles show GAPDH-HT (70 kDa) and likely the HT protein (30 kDa), respectively. Essentially the same results were obtained in an independent experiment. See also Fig. S2

Article Snippet: The mouse Lamp1 and Lamp2a cDNA clones in pCMV6-AC-GFP and the human LAMP2 , transcript variant A, cDNA clone in pCMV6 were obtained from ORIGENE (MG225631, MG222878, and RC221216, respectively).

Techniques: Activity Assay, Generated, Transfection, Fluorescence, Labeling, Staining, Expressing, Clone Assay, Stable Transfection, Control, Cell Culture, Western Blot