Journal: Journal of the American Society of Nephrology : JASN

Article Title: Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria

doi: 10.1681/ASN.2019090960

Figure Lengend Snippet: Glomerular cells of MLII but not of MLIII mice activate the integrated stress response (ISR) to alleviate proteostatic stress. (A) Immunoblot for the mTORC1 target S6 and its phosphorylated form p-S6 in MLII and MLIII mice demonstrates downregulation of total S6 in MLII and MLIII glomeruli, whereas the ratio of p-S6/S6 is not altered. Graphs show densitometric quantification normalized to β-actin of the same membrane, the relative levels to respective wild-type littermate controls are shown (mean±SEM, Mann–Whitney U test, n≥7, *P≤0.05). (B) High-resolution confocal microscopy for the transcription factor (TF) TFEB, which translocates to the nucleus to initiate the transcription of lysosomal genes in the setting of decreased mTORC1 activity. White arrows point toward podocyte (p) and endothelial cells (ec) with nuclear TFEB signal. (C) Activation of the ISR as a pathway to downregulate protein translation was evaluated by qPCR in MLIII and MLII glomeruli. Graph shows the relative gene of interest (GOI) expression to respective wild-type (WT) littermate controls (dashed line); internal controls used were 18S and glyceraldehyde-3-phosphate dehydrogenase (mean±SEM, Mann–Whitney U test, n≥5, *P≤0.05). (D) Immunoblot for the expression and activity (phosphorylation) levels of eIF2α as the core event of the ISR leading to a decrease in global protein synthesis and to the induction of selected genes such as the transcription factor ATF4 to promote cellular recovery in isolated glomeruli of MLIII and MLII mice. Graphs represent densitometric quantification normalized to β-actin of the same membrane, relative levels to respective wild-type littermate controls are shown (mean±SEM, Mann–Whitney U test, n≥7, *P≤0.05). High-resolution confocal microscopy exhibits expression of (E) activated p-eIF2α and its downstream target the transcription factor ATF4, (F) the activated ER stress sensor p-PERK, and (G) the transcription factors NRF1 and activated p-NRF2, which induce the transcription of proteasome genes. Podocyte slit membrane was visualized by staining for nephrin (red), DNA was counterstained with Hoechst (blue). Note the enhanced signal for p-PERK in MLII endothelial cells, for NRF1 and to a lesser extent for p-NRF2 in MLIII podocytes, of p-eIF2α in MLII podocytes and endothelial cells, and the marked ATF4 nuclear signal in MLII glomerular cells (white arrows). Rel., relative.

Article Snippet: Primary antibodies used for the study were rat anti–UCH-L1 (immunofluorescence [IF] microscopy 1:50; Western blot [WB] 1:250, self-made clone U104 17 ), guinea pig anti-nephrin (IF 1:100; Acris), mouse anti-ubiquitin (WB 1:250; Millipore), rabbit anti-ubiquitin (IF 1:300; Novus), rabbit anti–K48-polyubiquitin (WB 1:1000; Abcam), rabbit anti- β 5 (WB 1:1000; X. Wang, University of South Dakota), rabbit anti-LMP7 (WB 1:5000; E.K.), rabbit anti-Limp2 (WB 1:1000, IF 1:1000; P. Saftig, University of Kiel, Kiel, Germany), rabbit anti-Lamp2 (WB 1:1000, IF 1:400; Sigma-Aldrich), rabbit anti-LC3B (WB 1:1000, IF 1:50; Sigma-Aldrich), mouse anti– β -actin (WB 1:10,000; Sigma-Aldrich), goat anti–kidney injury molecule 1 (IF 1:1000; R&D Systems), rabbit anti–smooth muscle actin (IF 1:400; Abcam), rabbit anti–cleaved caspase 3 (IF 1:100; Cell Signaling), rhodamine wheat germ agglutinin (IF 1:400; Vector), rabbit anti-pS6 (IF 1:100, WB 1:1000; Cell Signaling), rabbit anti-S6 (WB 1:1000), rabbit anti–p-PERK (IF 1:100; Invitrogen), rabbit anti-ATF4 (WB 1:1000, IF 1:100; Bioss), rabbit anti-NRF1 (WB 1:1000, IF 1:100; Cell Signaling), rabbit anti–p(S40)-NRF2 (WB 1:1000, IF 1:50; Bioss), rabbit anti–eukaryotic translation initiation factor 2 α (anti-eIF2 α ; WB 1:1000), rabbit anti–p-eIF2 α (WB 1:1000, IF 1:50; Cell Signaling), and guinea pig anti-p62 (IF 1:100; Progen).

Techniques: Western Blot, Membrane, MANN-WHITNEY, Confocal Microscopy, Activity Assay, Activation Assay, Expressing, Isolation, Staining

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: RNA sequencing reveals that tomatidine modulates ATF4-dependent ER stress genes in pancreatic cancer cells Human and murine PDAC cell lines were treated for 40 h with 6.4 μg/mL tomatidine, RNA was isolated, and RNA sequencing was performed to analyze differences in gene regulation. N = 3 biological separate experiments. (A–D) (A) Heatmap of all genes and how they change in tomatidine-treated vs. untreated cells for Panc1 cells (FDR<0.05). Volcano plot highlighting ER stress-related genes in (B) Panc1 and (C) MT5 cells. (Upregulated genes on the right of the central axis and vice versa with higher fold change as we go away from the origin on y axis) (D) IPA upstream analysis of ATF4-related genes in Panc1 cells. (E) Heatmap elucidating targeting of ATF4-related genes in treated vs. untreated Panc1 cells. (Fold change: +2.5 to −1.5; FDR<0.03). (F) Top 10 pathways focusing on UPR, ATF4, and ER stress via Reactome analysis of the RNA-sequencing data of treated vs. untreated samples for Panc1 cells. (FDR< 0.03).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: RNA Sequencing, Isolation

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: ATF4 expression in PDAC (A) KM plotter was utilized to examine ATF4 expression in human PDAC specimens examining high vs. low expression compared to overall survival. (B) Data distribution elucidating significance of the overall survival curve prepared by KM Plotter. (C and D) (C) scRNA-seq datasets of pancreatic cancer tissue from metastatic patients were obtained from NIH dbGAP (accession phs002045.v1.p1) and (D) analyzed for ATF4 expression. (See also Table S1 ). (E) Pancreatic tumor tissue from KPC mice were stained by multiplex IF and imaged using Akoya Vectra Polaris and the Phenochart software to analyze ATF4 in multiple cellular compartments. Markers used to determine ATF4 (white), epithelial cells (CK19; red), stroma (αSMA; orange and PDGFRβ; Green), CD8 T+ cells (yellow), and macrophages (F4/80; turquoise). Scale bar = 100 μm.

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Staining, Multiplex Assay, Software

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine inhibits ATF4-dependent signaling in PDAC (A and B) (A) MiaPaca-2 tumor cells were treated with tomatidine for 72 h and cell lysates were immunoblotted for ATF4, 4EBP1, and phospho-4EBP1(p-4EBP1) protein expression and (B) p-4EBP1/4EBP1 levels quantified by densitometry. (C) Immunofluorescence (IF) was performed on vehicle (DMSO) or tomatidine-treated Panc1 cells to track ATF4 (FITC-Green) translocation from nucleus (DAPI-Blue) to cytoplasm. Scale bar = 50 μm. (D–G) (D) Nuclear to cytoplasmic translocation was quantified. Panc1 cells were treated with vehicle (DMSO) or tomatidine for 40 h and ATF4 transcriptional activity was analyzed by chromatin immunoprecipitation (ChIP) qPCR evaluating binding of ATF4 to the downstream promoter regions of (E) eIF4EBP1 (F) CHOP- B site and (G) ASNS. Data are reported as the means + SEMs. n = 3 or more independent biological replicates (4B, One-way ANOVA with Tukey’s test for pairwise comparisons was used to analyze the data; 4D-G, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Immunofluorescence, Translocation Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: In vivo tomatidine treatment inhibits pancreatic tumor growth (A–C) (A) MT5 tumor-bearing C57BL/6 mice (5 mice/group) were treated with 5 mg/kg daily i.p. injections of tomatidine or vehicle control (40% HPBCD) and monitored for tumor growth. (n = 5/group). RNA isolated from the tumor tissues of MT5 tumor-bearing C57BL/6 mice treated with vehicle or 5 mg/kg daily i.p. injections of tomatidine were assessed for (B) ATF4 and (C) eIF4EBP1 expression via qPCR. Data are reported as the means + SEMs. n = 5 mice per group. (5A, mixed between-within subjects ANOVA shows a significant interaction between days of treatment and group; 5B-C, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: In Vivo, Control, Isolation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine mediated inhibition of ATF4 signaling can increase sensitivity to ferroptotic cell death in PDAC (A) Panc-1 tumor cells were treated with tomatidine (6.4 μg/mL) and analyzed by RNA sequencing. Ingenuity pathway analysis of the regulated genes suggested ferroptosis as a top hit for tomatidine-treated cells. (B–D) (B) Pancreatic cancer cells were treated with vehicle (DMSO), erastin (to induce ferroptosis), ferrostatin-1 (to inhibit ferroptosis), tomatidine, or in different combinations and lipid peroxidation of (C) Panc-1 and (D) MiaPaca-2 was analyzed by flow cytometry using Bodipy-11. (E) Panc-1 and MiaPaca-2 cells treated with vehicle (DMSO) or tomatidine and lysates collected after 24 h were immunoblot for GPX4 expression. (F) Panc1 cells were plated overnight and treated with tomatidine for 6 h and then assayed by Seahorse assay to analyze mitochondrial fitness. (G) Schematic showing how tomatidine can regulate ATF4-dependent signaling to induce ferroptosis in pancreatic cancer. Data are reported as the means + SEMs. n = 3 or more independent biological replicates. (7C-D, Two-tailed independent student’s test was used to analyze the data; 7F, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05, ∗∗p < 0.007).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Inhibition, RNA Sequencing, Flow Cytometry, Western Blot, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet:

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Control, Recombinant, Viability Assay, Luciferase, ChIP-qPCR, Gene Expression, Software

Journal: Antioxidants

Article Title: Curculigoside Attenuates Endoplasmic Reticulum Stress-Induced Epithelial Cell and Fibroblast Senescence by Regulating the SIRT1-P300 Signaling Pathway

doi: 10.3390/antiox13040420

Figure Lengend Snippet: The information of the primary antibodies.

Article Snippet: Anti-ATF4 Polyclonal antibody , Proteintech , 1:1000.

Techniques: Recombinant

Journal: Antioxidants

Article Title: Curculigoside Attenuates Endoplasmic Reticulum Stress-Induced Epithelial Cell and Fibroblast Senescence by Regulating the SIRT1-P300 Signaling Pathway

doi: 10.3390/antiox13040420

Figure Lengend Snippet: CCG alleviated BLM-induced pulmonary senescence. ( A ) Immunofluorescence staining showed the colocalization of the senescence marker P21 and the fibroblast marker α-SMA. Red represents P21, green represents α-SMA, and blue represents DAPI. Scale bars represent 100 μm. ( B ) Immunofluorescence staining showed the colocalization of the senescence marker P21 and the AEC marker SPC. Red represents P21, green represents SPC, and blue represents DAPI. Scale bars represent 100 μm. ( C , D ) Expression levels of senescence markers P53, P21, and P16 detected via WB. ( E , F ) Expression levels of ERS markers ATF4, CHOP, and BIP detected via WB. Data represent means ± standard deviation, with each experiment independently repeated at least three times. (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Anti-ATF4 Polyclonal antibody , Proteintech , 1:1000.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Standard Deviation

Journal: Antioxidants

Article Title: Curculigoside Attenuates Endoplasmic Reticulum Stress-Induced Epithelial Cell and Fibroblast Senescence by Regulating the SIRT1-P300 Signaling Pathway

doi: 10.3390/antiox13040420

Figure Lengend Snippet: CCG attenuated ROS-mediated ERS in AECs. ( A ) H2DCFDA probe labeling of ROS (green) in AECs. Scale bars represent 100 μm. ( B ) Fluo-3AM probe (green) reflecting intracellular calcium influx in AECs. Scale bars represent 100 μm. ( C ) Flow cytometry detection of ROS levels in AECs. ( D ) Flow cytometry detection of intracellular calcium influx in AECs. ( E , F ) Western blot analysis of expression levels of ERS markers ATF4, CHOP, and BIP. Data represent means ± standard deviation, with each experiment independently repeated at least three times. (* p < 0.05, and *** p < 0.001).

Article Snippet: Anti-ATF4 Polyclonal antibody , Proteintech , 1:1000.

Techniques: Labeling, Flow Cytometry, Western Blot, Expressing, Standard Deviation

Journal: Antioxidants

Article Title: Curculigoside Attenuates Endoplasmic Reticulum Stress-Induced Epithelial Cell and Fibroblast Senescence by Regulating the SIRT1-P300 Signaling Pathway

doi: 10.3390/antiox13040420

Figure Lengend Snippet: CCG attenuated ROS-mediated ERS in fibroblasts. ( A ) H2DCFDA probe labeling of ROS (green) in 5–10th passage fibroblasts. Scale bars represent 100 μm. ( B ) Fluo-3AM probe (green) reflecting intracellular calcium influx in 5–10th passage fibroblasts. Scale bars represent 100 μm. ( C , D ) Western blot analysis of expression levels of ERS markers ATF4, CHOP, and BIP in 5–10th passage fibroblasts after treatment with 10 μM CCG. ( E , F ) Western blot analysis of expression levels of ERS markers ATF4, CHOP, and BIP in 10th passage fibroblasts after treatment with 1, 3, and 10 μM CCG. Data represent means ± standard deviation, with each experiment independently repeated at least three times. (** p < 0.01, and *** p < 0.001).

Article Snippet: Anti-ATF4 Polyclonal antibody , Proteintech , 1:1000.

Techniques: Labeling, Western Blot, Expressing, Standard Deviation

Journal: Antioxidants

Article Title: Curculigoside Attenuates Endoplasmic Reticulum Stress-Induced Epithelial Cell and Fibroblast Senescence by Regulating the SIRT1-P300 Signaling Pathway

doi: 10.3390/antiox13040420

Figure Lengend Snippet: SIRT1 mediated the effect of CCG on ROS-mediated ERS in AECs. ( A ) H2DCFCDA probe labeling of ROS (green) in AECs. Scale bars represent 100 μm. ( B ) Fluo-3AM probe (green) reflecting intracellular calcium influx in AECs. Scale bars represent 100 μm. ( C ) Flow cytometry analysis of ROS levels in AECs. ( D ) Flow cytometry analysis of intracellular calcium influx in AECs. ( E , F ) WB analysis of expression levels of ERS markers ATF4, CHOP, and BIP. Data represent means ± standard deviation, with each experiment independently repeated at least three times. (*** p < 0.001).

Article Snippet: Anti-ATF4 Polyclonal antibody , Proteintech , 1:1000.

Techniques: Labeling, Flow Cytometry, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation independently of the p97 ATPase

doi: 10.1101/2022.12.16.520763

Figure Lengend Snippet: (A) Immunoblot of HFT wildtype and UBXN1 KO cells treated with 1.5 mM dithiothreitol (DTT) for the indicated timepoints (0-8 hours) to visualize downstream PERK targets. (B) Ratio (UBXN1 KO/wildtype) of the band intensity quantifications of BiP, ATF4, and peIF2 α at the 8-hour timepoint corresponding to . ( n = three biologically independent samples). We only provide the 8-hour quantification as the signal is not present in wildtype cells in earlier timepoints for accurate quantification. (C) Immunoblot of HFT wildtype and UBXN1 KO cells co-treated with 1.5 mM DTT and 1 µM Bortezomib (BTZ) to visualize full-length and cleaved ATF6. (D) ATF6 activation was measured by band intensity quantification and calculation of the percentage of cleaved ATF6 to total ATF6. The ratio of the percentage of ATF6 activation in UBXN1 KO cells to wildtype is reported. ( n = four biologically independent samples). (E) Representative immunofluorescent image of IRE1 α clustering in a stable, doxycycline inducible HFT cell line expressing GFP-tagged IRE1 α . UBXN1 was depleted by siRNA transfection and cells were treated with 2.5 µM tunicamycin (Tu) for 4 hours to induce IRE1 α clustering (Scale bar: 10 µm). (F) Quantification corresponding to reporting the percent of cells with GFP-IRE1 α foci as well as the number of GFP-IRE1 α foci per cell ( n = three biologically independent samples). (G) Transcript levels of xbp1s and total xbp1 in HFT wildtype and UBXN1 KO cells quantified by real-time PCR. Cells were treated with 10 nM thapsigargin (Tg) for 4 hours as indicated. ( n = three biologically independent samples). Data are means ± SEM (*, **, ***, **** where P < 0.05, 0.01, 0.001, and 0.0001, respectively.) Unpaired two-tailed t test (B, D) or One-way ANOVA with Tukey’s multiple comparisons test (F, G) .

Article Snippet: The rabbit UBXN1 (16135-1-AP 1:7000 dilution), p97 (10736-1-AP), BAG6 (26417-1-AP), ATF6 (24169-1-AP), Sec61 β (51020-2-AP 1:3000 dilution), Aconitase 2 (11134-1-AP), calnexin (10427-2-AP), CYC1 (10242-1-AP), and G3BP1 (13057-2-AP) antibodies were obtained from Proteintech Inc; rabbit BiP (3177 dilution), peIF2 α (3398 1:500 dilution), and MCM3 (4012S) antibodies were obtained from Cell Signaling Technology; mouse PCNA (sc-56 1:3000 dilution), ATF4 (sc-390063 1:500 dilution), Tom20 (sc-17764), ubiquitin (P4D1; sc8017), β -actin (sc-69879 1:3000 dilution), GAPDH (sc-47724 1:3000 dilution), c-myc (sc-40 1:3000 dilution), Sec61 α (sc-393182 1:500 dilution), and TRAP α (sc-373916) were from Santa Cruz Biotechnology; mouse ubiquitin FK2 (04-263 1:100 dilution) for immunofluorescence and Anti-Puromycin, clone 12D10 (MABE343 1:3000 dilution) was from EMD Millipore; mouse FLAG-M2 antibody (F3165 1:1000 dilution) was from Sigma Aldrich.

Techniques: Western Blot, Activation Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: bioRxiv

Article Title: UBXN1 maintains ER proteostasis and represses UPR activation by modulating translation independently of the p97 ATPase

doi: 10.1101/2022.12.16.520763

Figure Lengend Snippet: (A) Immunoblot of BiP and ATF4 expression levels in HFT wildtype cells treated with 1 µM thapsigargin (Tg), 5 µM of the ATP-competitive p97 inhibitor CB-5083, or both for the indicated timepoints (0-6 hours). (B-C) Band intensity quantifications of BiP (B) and ATF4 (C) corresponding to reporting the fold change compared to the untreated condition ( n = three biologically independent samples) . (D) Immunoblot of HFT wildtype cells depleted of BAG6 with siRNA for 48 hours before treatment with 1.5 mM DTT for the indicated timepoints (0-8 hours). (E) Band intensity quantifications of BiP and ATF4 corresponding to . at the 8-hour timepoint. ( n = four biologically independent experiments). (F) Transcript levels of xbp1s and total xbp1 in HEK-293T wildtype and UBXN1 KO cells quantified by real-time PCR. Cells were treated with 10 nM thapsigargin (Tg) for 4 hours as indicated ( n = three biologically independent samples). (G) Immunofluorescent staining of ubiquitin (FK2) and nuclei (Hoechst dye) in HFT wildtype and UBXN1 KO cells. Cells were treated with 1 µM of the proteasome inhibitor bortezomib (BTZ), Tunicamycin (Tu), or Thapsigargin (Tg) for 8 hours or 1 µM BTZ, 500 nM Tu, or 500 nM Tg for 18 hours. (H) Immunoblot assessing total ubiquitin levels corresponding to . Data are means ± SEM (*, **, ***, **** where P < 0.05, 0.01, 0.001, and 0.0001, respectively.) One-way ANOVA with Dunnetts’s multiple comparisons test (B and C). Unpaired two-tailed t test (E). One-way ANOVA with Tukey’s multiple comparisons test (F) .

Article Snippet: The rabbit UBXN1 (16135-1-AP 1:7000 dilution), p97 (10736-1-AP), BAG6 (26417-1-AP), ATF6 (24169-1-AP), Sec61 β (51020-2-AP 1:3000 dilution), Aconitase 2 (11134-1-AP), calnexin (10427-2-AP), CYC1 (10242-1-AP), and G3BP1 (13057-2-AP) antibodies were obtained from Proteintech Inc; rabbit BiP (3177 dilution), peIF2 α (3398 1:500 dilution), and MCM3 (4012S) antibodies were obtained from Cell Signaling Technology; mouse PCNA (sc-56 1:3000 dilution), ATF4 (sc-390063 1:500 dilution), Tom20 (sc-17764), ubiquitin (P4D1; sc8017), β -actin (sc-69879 1:3000 dilution), GAPDH (sc-47724 1:3000 dilution), c-myc (sc-40 1:3000 dilution), Sec61 α (sc-393182 1:500 dilution), and TRAP α (sc-373916) were from Santa Cruz Biotechnology; mouse ubiquitin FK2 (04-263 1:100 dilution) for immunofluorescence and Anti-Puromycin, clone 12D10 (MABE343 1:3000 dilution) was from EMD Millipore; mouse FLAG-M2 antibody (F3165 1:1000 dilution) was from Sigma Aldrich.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Staining, Ubiquitin Proteomics, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: Sestrin2 protects against lethal sepsis by suppressing the pyroptosis of dendritic cells

doi: 10.1007/s00018-021-03970-z

Figure Lengend Snippet: ERS induces NLRP3/CASP-1-dependent pyroptosis and SESN2 expression in DCs. A, Western blotting was used to measure the expression of ERS markers (GRP78, ATF4, and CHOP) in DCs at 6 h, 12 h, 24 h, 48 h, or 72 h post CLP. B, Primary DCs were treated with 2 μg/ml TM for 24 h, and Western blotting was used to evaluate the expression of SESN2, NLRP3 inflammasome components, and ERS markers. C, Assays were performed 24 h after the mice were intraperitoneally administered physiological saline or TM (2 mg/kg body weight). Immunoblots of SESN2, NLRP3 inflammasome components, and ERS markers are shown. The values are protein levels relative to the β-actin levels. The data are representative of 3 independent experiments (5 mice were pooled together for each independent experiment). D, DC pyroptosis was assessed by flow cytometry after treatment with 2 μg/ml TM for 24 h in vitro, DCs were labeled with active CASP-1 (FAM-FLICA) and 7-AAD. Representative flow cytometry plots are shown on the left, and quantitative analysis of FLICA + 7AAD + DCs is shown on the right ( n = 4). Statistical significance: * P < 0.05 versus the control group

Article Snippet: A mouse anti-mouse NLRP3/NALP3 monoclonal antibody was purchased from AdipoGen (San Diego, CA), and a mouse anti-mouse ATF4 monoclonal antibody was purchased from Proteintech Group (Rosemont, IL).

Techniques: Expressing, Western Blot, Saline, Flow Cytometry, In Vitro, Labeling, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Sestrin2 protects against lethal sepsis by suppressing the pyroptosis of dendritic cells

doi: 10.1007/s00018-021-03970-z

Figure Lengend Snippet: SESN2 deficiency exacerbates sepsis-induced ERS. WT mice and SESN2 −/− mice were subjected to sham surgery or CLP. A, SESN2 expression and the ER in DCs were examined by laser scanning confocal microscopy 24 h after CLP. DyLight 488 (green)-labeled SESN2 protein, ER tracker (red), and DAPI-stained nuclei (blue) were visualized using indirect immunofluorescence and confocal microscopy (× 600, × 1200). B, Immunoblot analysis of GRP78, ATF4, and CHOP expression in DCs from WT and SESN2 −/− mice at 24 h after CLP. C, Morphological alterations in the ER in DCs from WT and SESN2 −/− mice were measured by immunofluorescence staining at 24 h after CLP (× 600, × 1200). D, ATF4 (DyLight 594, red) expression in DCs was observed by using indirect immunofluorescence and confocal microscopy, and DAPI-stained nuclei (blue) in DCs from WT and SESN2 −/− mice were visualized at 24 h after CLP (× 600, × 1200). E, Immunoblot analysis of ATF4 expression in the cytosol and nucleus. β-actin served as a control for cytosolic proteins. Histone3 served as a control for nuclear proteins. The data are presented as the mean ± SD of at least three independent experiments. Statistical significance: * P < 0.05 versus the sham group

Article Snippet: A mouse anti-mouse NLRP3/NALP3 monoclonal antibody was purchased from AdipoGen (San Diego, CA), and a mouse anti-mouse ATF4 monoclonal antibody was purchased from Proteintech Group (Rosemont, IL).

Techniques: Expressing, Confocal Microscopy, Labeling, Staining, Immunofluorescence, Western Blot, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Sestrin2 protects against lethal sepsis by suppressing the pyroptosis of dendritic cells

doi: 10.1007/s00018-021-03970-z

Figure Lengend Snippet: The PERK–ATF4–CHOP pathway is critically involved in the inhibitory effect of SESN2 on DC pyroptosis. DCs were isolated from the spleens of WT and SESN2 −/− mice treated with or without salubrinal at 24 h after sham surgery or CLP. A, DC pyroptosis was assessed by flow cytometry, and DCs were labeled with active CASP-1 (FAM-FLICA) and 7-AAD. Representative flow cytometry plots are shown on the left, and quantitative analyses of FLICA + 7AAD + DCs are shown on the right ( n = 6). B, Immunoblot analysis of the expression of inflammasome markers, including CASP-1, GSDMD, NLRP3, and ASC and signaling molecules, including PERK, ATF4, CHOP, and GRP78. β-actin served as an internal control. C, Representative confocal immunofluorescence microscopy images of NLRP3 colocalized with ASC. DyLight 488 (green)-labeled ASC protein, DyLight 594 (red)-labeled NLRP3 protein, and DAPI (blue)-stained nuclei are shown. The images are representative of six independent samples from each group. The data are presented as the mean ± SD of at least three independent experiments. Statistical significance: * P < 0.05 versus the control group. D, Kaplan–Meier survival curve analysis was performed for mice with CLP-induced sepsis for 7 days (n = 12). Mice in the sham group underwent surgery without CLP and received 4 ml/kg PBS at 1 h after the operation, mice in the CLP group received 4 ml/kg PBS at 1 h after CLP, and mice in the CLP + salubrinal group received 20 mg/kg salubrinal at 1 h after CLP

Article Snippet: A mouse anti-mouse NLRP3/NALP3 monoclonal antibody was purchased from AdipoGen (San Diego, CA), and a mouse anti-mouse ATF4 monoclonal antibody was purchased from Proteintech Group (Rosemont, IL).

Techniques: Isolation, Flow Cytometry, Labeling, Western Blot, Expressing, Control, Immunofluorescence, Microscopy, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Sestrin2 protects against lethal sepsis by suppressing the pyroptosis of dendritic cells

doi: 10.1007/s00018-021-03970-z

Figure Lengend Snippet: Basic schematic of the mechanism by which SESN2 inhibits pyroptosis and prolongs NLRP3 inflammasome activation by preserving ERS homeostasis. In splenic DCs, SESN2 expression was enhanced after CLP or stimulation with LPS via the PERK–ATF4–CHOP pathway. Sepsis per se induced severe latent ERS, and hyperactivation of ATF4 resulted in obvious elevation of CHOP expression, which might act as a potentiating step. Furthermore, CHOP activated the NLRP3 inflammasome and promoted the activation of pro-CASP-1 to CASP-1. Upon activation, CASP-1 cleaved the N-terminal fragment of GSDMD, and then GSDMD-N oligomerized in the plasma membrane and formed pores, resulting in the release of cellular contents and pyroptosis. Moreover, CASP-1 activation led to the maturation of IL-1β and IL-18 and the release of cytokines (IL-6, HMGB1, TNF-α, etc.), thereby contributing to the inflammatory response, immune dysfunction, and even cell death. Upregulation of SESN2 might reduce protein translation, and attenuate ERS, and subsequently suppress NLRP3 inflammasome activation through the PERK–ATF4–CHOP pathway, controlling the excessive inflammatory response and pyroptosis secondary to septic challenge

Article Snippet: A mouse anti-mouse NLRP3/NALP3 monoclonal antibody was purchased from AdipoGen (San Diego, CA), and a mouse anti-mouse ATF4 monoclonal antibody was purchased from Proteintech Group (Rosemont, IL).

Techniques: Activation Assay, Preserving, Expressing, Clinical Proteomics, Membrane

Journal: Molecular Medicine Reports

Article Title: Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

doi: 10.3892/mmr.2025.13532

Figure Lengend Snippet: Primer sequences.

Article Snippet: ATF4 , Cat. no. BM5179, rabbit mAb (Wuhan Boster Biological Technology, Ltd.) , WB 1:1,000.

Techniques: Sequencing

Journal: Molecular Medicine Reports

Article Title: Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

doi: 10.3892/mmr.2025.13532

Figure Lengend Snippet: Antibodies used for immunofluorescence and WB analyses.

Article Snippet: ATF4 , Cat. no. BM5179, rabbit mAb (Wuhan Boster Biological Technology, Ltd.) , WB 1:1,000.

Techniques: Immunofluorescence

Journal: Molecular Medicine Reports

Article Title: Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

doi: 10.3892/mmr.2025.13532

Figure Lengend Snippet: Detection of mRNA and protein expression levels of ER stress-related genes and proteins in ASCs induced by TG. (A) Reverse transcription-quantitative PCR analysis of the expression of ER-stress-related genes in ASCs in each group (one-way ANOVA followed by Tukey's multiple comparison post hoc analysis; *P<0.05; **P<0.01; ***P<0.001; n=3). (B) Representative western blots. (C) Western blot analysis of ER stress-related proteins in each group (one-way ANOVA followed by Tukey's post hoc analysis; **P<0.01; ***P<0.001; n=3-5). 1: Untreated group; 2: DMEM + complete medium group; 3: TG + complete medium group; 4: TG group. ER, endoplasmic reticulum; ASCs, astrocytes; TG, thapsigargin; GRP78, glucose regulated protein 78; ATF4, activating transcription factor 4; DMEM, Dulbecco's modified Eagle's medium.

Article Snippet: ATF4 , Cat. no. BM5179, rabbit mAb (Wuhan Boster Biological Technology, Ltd.) , WB 1:1,000.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison, Western Blot, Modification

Journal: Molecular Medicine Reports

Article Title: Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

doi: 10.3892/mmr.2025.13532

Figure Lengend Snippet: Verification of propagation of ER stress from ASCs to neurons. (A) Optical microscopy was performed to observe the morphological changes of PC12 cells in each group, scale bar, 50 µm. (B) Reverse transcription-quantitative PCR analysis of ER stress-related mRNA expression in PC12 cells. (C) Representative western blots of the ER stress-associated proteins in PC12 cells in each group. (D) Semi-quantitative analysis of ER stress-related proteins levels in PC12 cells in each group (one-way ANOVA followed by Tukey's post hoc analysis; **P<0.01; ***P<0.001; n=3). (E) Immunofluorescence experiment of PDI fluorescence intensity in PC12 cells. (F) Apoptosis probe analysis of PC12 cell in each group, scale bar, 100 µm. 1: UT group; 2: UT-ACM group; 3: TG-ACM group; 4: TG group. ER, endoplasmic reticulum; ASCs, astrocytes; UT, untreated; ACM, astrocyte conditioned medium; PDI, protein disulfide isomerase; TG, thapsigargin; GRP78, glucose regulated protein 78; ATF4, activating transcription factor 4.

Article Snippet: ATF4 , Cat. no. BM5179, rabbit mAb (Wuhan Boster Biological Technology, Ltd.) , WB 1:1,000.

Techniques: Microscopy, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Fluorescence

Journal: Molecular Medicine Reports

Article Title: Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

doi: 10.3892/mmr.2025.13532

Figure Lengend Snippet: Isolation and preparation of ACM-F and ACM-R components, and detection of mRNA expression levels of ER stress-related genes in PC12 cells. (A) Full-wavelength spectrophotometry was performed to measure the residual amount of TG in the supernatant. (B) Ultracentrifugation to separate the components of the ACM-R and ACM-F. (C) Coomassie brilliant blue staining to detect the molecular weight of the ACM-R and ACM-F. (D) Reverse transcription-quantitative PCR analysis was performed to detect ER stress-related mRNA expression in PC12 cells in each group (one-way ANOVA followed by Tukey's post hoc analysis; ***P<0.001; n=3). 1: UT group; 2: UT-ACM group; 3: UT-ACM-F group; 4: UT-ACM-R group; 5: TG-ACM group; 6: TG-ACM-F group; 7: TG-ACM-R group; 8: TG group. ACM, astrocyte conditioned medium; UT, untreated; ACM-F, ACM filtrate; ER, endoplasmic reticulum; TG, thapsigargin; ACM-R, ACM-F residue; GRP78, glucose regulated protein 78; ATF4, activating transcription factor 4.

Article Snippet: ATF4 , Cat. no. BM5179, rabbit mAb (Wuhan Boster Biological Technology, Ltd.) , WB 1:1,000.

Techniques: Isolation, Expressing, Spectrophotometry, Staining, Molecular Weight, Reverse Transcription, Real-time Polymerase Chain Reaction, Residue

Journal: Molecular Medicine Reports

Article Title: Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

doi: 10.3892/mmr.2025.13532

Figure Lengend Snippet: Determination of ACM vesicle size and detection of mRNA expression levels of ER stress-related genes in PC12 cells induced by ACM. (A) Nanoparticle size analysis of ACM-vesicles. (B) Reverse transcription-quantitative PCR analysis of the mRNA expression levels of ER stress-related genes in PC12 cells in each group (one-way ANOVA followed by Tukey's post hoc analysis; **P<0.01; ***P<0.001; n=3). 1: UT group; 2: UT-ACM group; 3: UT-ACM-(no) vesicles group; 4: UT-ACM-vesicles group; 5: TG-ACM group; 6: TG-ACM-(no) vesicles group; 7: TG-ACM-vesicles group; 8: TG group. ACM, astrocyte conditioned medium; ER, endoplasmic reticulum, ER; UT, untreated; TG, thapsigargin; GRP78, glucose regulated protein 78; ATF4, activating transcription factor 4.

Article Snippet: ATF4 , Cat. no. BM5179, rabbit mAb (Wuhan Boster Biological Technology, Ltd.) , WB 1:1,000.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Preliminary exploration of endoplasmic reticulum stress transmission in astrocytes and neurons, and its mediators

doi: 10.3892/mmr.2025.13532

Figure Lengend Snippet: Effect of supplementing Vc in the ACM on ER stress propagation. (A) Morphological changes of PC12 cells in each group observed under a light microscope; scale bar, 50 µm. (B) MTT assay was performed to detect changes in PC12 cell viability in each group (one-way ANOVA followed by Tukey's post hoc analysis; n=5). (C) Representative western blots of ER stress-related proteins in PC12 cells in each group. (D) Semi-quantitative analysis of ER stress-related protein levels in PC12 cells in each group (one-way ANOVA followed by Tukey's multiple comparison; n=3). (E) Immunofluorescence experiment to detect PDI fluorescence intensity of PC12 cells; scale bar, 100 µm. (F) Full-wavelength spectrophotometry was performed to draw the Vc standard curve. (G) Vc residue in the supernatant of each group was measured using a spectrophotometer. *P<0.05, **P<0.01 and ***P<0.001. 1: UT group; 2: UT-ACM group; 3: TG-ACM group; 4: TG-ACM + Vc group; 5: TG group. ER, endoplasmic reticulum; PDI, protein disulfide isomerase; OD, optical density; UT, untreated; ACM, astrocyte conditioned medium; TG, thapsigargin; GRP78, glucose regulated protein 78; ATF4, activating transcription factor 4.

Article Snippet: ATF4 , Cat. no. BM5179, rabbit mAb (Wuhan Boster Biological Technology, Ltd.) , WB 1:1,000.

Techniques: Light Microscopy, MTT Assay, Western Blot, Comparison, Immunofluorescence, Fluorescence, Spectrophotometry, Residue

Journal: bioRxiv

Article Title: Targeting the cell and non-cell autonomous regulation of 47S synthesis by GCN2 in colon cancer

doi: 10.1101/2023.02.08.527626

Figure Lengend Snippet: A. Gene Ontology enrichment analysis on TCGA database highlights an upregulation of ribosomal biogenesis processes in COAD compared to normal tissue. Top enrichment GO terms in COAD tumors versus normal tissues are shown and significance is expressed as -Log10 adjusted p-value. B. Dysregulated expression of riboproteins (RP) is higher in COAD compared to the normal tissue. TCGA COAD data, Mann-Whitney-test, (*** p < 0.001) C. The amino acid deprivation gene signature is enriched in COAD tumors with high dysregulated RP expression. TCGA COAD data, Mann-Whitney-test, (** p < 0.01) D. ATF4 gene signature is higher in colon tumors compared to normal tissue. TCGA COAD data, Mann-Whitney-test, (*** p < 0.001) E. ATF4 gene signature is enriched in COAD with high dysregulation of RP expression. TCGA COAD data, Mann-Whitney-test (* p < 0.05)

Article Snippet: Regarding RNA interference experiments, HCT116 cells were seeded onto 6-well plates (2.5 × 10 5 cells per well) and concomitantly transfected with non-targeting siRNA (sc-37007) or human GCN2 siRNA (sc-45644) or human ATF4 siRNA (sc-35112) or human MetRS siRNA (sc-75775) (Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Targeting the cell and non-cell autonomous regulation of 47S synthesis by GCN2 in colon cancer

doi: 10.1101/2023.02.08.527626

Figure Lengend Snippet: A. Cell proliferation assessed by the confluency index of HCT116 cells treated or not with GCN2i for 96 h. Data are expressed as mean +/-s.e.m of independent experiments (n = 3). Unpaired two-tailed t-test with p-value (** p < 0.01, *** p < 0.001). B. Cell mass was assessed every day over the indicated period of time in HCT116 cells treated or not with GCN2i by SRB assay. A daily refreshment of the medium (indicated by the black rounded arrow) was performed to ensure a full supply of nutrients over the time course of the experiment. Data are expressed as mean +/-s.e.m of independent experiments (n = 3). Unpaired two-tailed t-test with p-value (** p < 0.01). C. Relative number of HCT116 cells transfected either with a siRNA control (CTRL) or against GCN2 (GCN2) following 4 days of culture. Medium were daily refreshed (black rounded arrow) to prevent nutrients exhaustion. Data are expressed as mean +/-s.e.m of 3 independent experiments. Unpaired two-tailed t-test with p-value (** p < 0.01). D. Cell mass measurement of patient-derived primary cells cultured in 2D conditions and treated with DMSO or GCN2i for 48 h. Media were refreshed daily (black rounded arrow). E. Spheroid formation assays of HCT116 treated with DMSO or GCN2i. ATP content was measured after 7 days of treatment. Treatment was initiated concomitantly to plating. Data are expressed as mean +/-s.e.m of 3 independent experiments. Unpaired two-tailed t-test with p-value (** p < 0.01). F. Western blot analysis of P-eIF2a, ATF4 and CHOP protein amounts in HCT116 starved for leucine for 8h in the presence of ISRIB (200 nM) or not. G. Measurements of cell mass in HCT116 cells treated with ISRIB (200 nM) for 48 h. Medium were daily refreshed (black rounded arrow) to ensure complete nutrients supply. Data are expressed as mean +/-s.e.m of independent experiments (n = 5). Unpaired two-tailed t-test. H. Western blot analysis of ATF4 protein levels in HCT116 cells transfected either with a siRNA control (siCtrl) or against ATF4 (siATF4) following 2 days of culture with siCTRL and siATF4 upon 8 h of amino acid deprivation (leucine starvation). I. Cell proliferation assessed by the confluency index of HCT116 cells transfected with a siCtrl and siATF4 was monitored over 48 h. Data are expressed as mean +/-s.e.m of independent experiments (n = 3). Unpaired two-tailed t-test with p-value (* p < 0.05).

Article Snippet: Regarding RNA interference experiments, HCT116 cells were seeded onto 6-well plates (2.5 × 10 5 cells per well) and concomitantly transfected with non-targeting siRNA (sc-37007) or human GCN2 siRNA (sc-45644) or human ATF4 siRNA (sc-35112) or human MetRS siRNA (sc-75775) (Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Two Tailed Test, Sulforhodamine B Assay, Transfection, Control, Mass Measurement, Derivative Assay, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Targeting the cell and non-cell autonomous regulation of 47S synthesis by GCN2 in colon cancer

doi: 10.1101/2023.02.08.527626

Figure Lengend Snippet: A. Gene set enrichment analysis in HCT116 cells silenced for GCN2 compared to the control. Top enrichment GO terms in siGCN2 transfected cells versus siCtrl are shown and significance is expressed as -Log10 adjusted p-value. B. Amino acid profiling of HCT116 cells transfected with a siRNA control or against GCN2. C. Assessment of protein synthesis rate in HCT116 cells treated for 24 h with GCN2i. D. Western blot analysis of autophagic markers, LC3-I, LC3-II and p62, in HCT116 cells treated with GCN2i for the indicated period of time. E. Autophagic flux analysis using HCT116 cells stably expressing the GFP-LC3-RFP-LC3ΔG construct and treated with GCN2i for 48 h. Cleavage of GFP-LC3 by autophagy released the RFP-LC3 as an internal control, thus reduction of the GFP/RFP ratio illustrates induction of the autophagy flux. Data are expressed as the mean of quantification +/-s.e.m. of independent experiments (n = 3). Unpaired two-tailed t test with p-value (* p < 0.05) F. Cell mass was measured in GCN2i-treated HCT116 cells after 48 h in combination with chloroquine. Data are expressed relative to the vehicle as mean +/-s.e.m of independent experiments (n = 7). Unpaired two-tailed t-test with p-value (*** p < 0.001). G. Western blot analysis of mTORC1 pathway markers (P-S6, P-4EBP1 and P-ULK1) amounts in HCT116 cells following 24 h of GCN2i treatment. H. Immunohistochemical staining against P-S6 in HCT116 spheroids treated for 3 days with DMSO or GCN2i. Quantification of the IHC staining is provided on the right panel. Data are expressed as mean +/-s.e.m of independent experiments (n = 3). Unpaired two-tailed t-test with p-value (* p < 0.05). I. Gene set enrichment analysis of COAD patients with low GCN2 expression (25%) compared to the rest of the COAD cohort (TCGA data).

Article Snippet: Regarding RNA interference experiments, HCT116 cells were seeded onto 6-well plates (2.5 × 10 5 cells per well) and concomitantly transfected with non-targeting siRNA (sc-37007) or human GCN2 siRNA (sc-45644) or human ATF4 siRNA (sc-35112) or human MetRS siRNA (sc-75775) (Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Control, Transfection, Western Blot, Stable Transfection, Expressing, Construct, Two Tailed Test, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Targeting the cell and non-cell autonomous regulation of 47S synthesis by GCN2 in colon cancer

doi: 10.1101/2023.02.08.527626

Figure Lengend Snippet: A. Time course analysis of cell cycle progression by monitoring HCT116 FUCCI cells upon DMSO or GCN2i. Data are expressed relative to the vehicle as mean +/-s.e.m of independent experiments (n = 3). B. Fibrillarin localization assessed by immunofluorescence in HCT116 cells treated with DMSO or GCN2i for 4 hours. Nuclei were stained by Hoechst solution. C. Time course analysis of cellular rRNAs by northern blotting following GCN2i treatment. D. Cell proliferation assessed by the confluency index of HCT116 cells treated or not with GCN2i for 72 h. Data are expressed as mean +/-s.e.m of independent experiments (n = 3). Unpaired two-tailed t-test with p-value (** p < 0.01, *** p < 0.001). E. Fibrillarin localization assessed by immunofluorescence in HCT116 cells, 48 hours after transfection with a siRNA control (Ctrl) or against MetRS. Nuclei were stained by Hoechst solution. F. Western blot analysis of MetRS amounts in the cytoplasmic or nuclear fractions upon 2 hours of GCN2i and GCNiB treatments. Histone H3 and tubulin are provided as loading controls for respectively nuclear and cytoplasmic samples.

Article Snippet: Regarding RNA interference experiments, HCT116 cells were seeded onto 6-well plates (2.5 × 10 5 cells per well) and concomitantly transfected with non-targeting siRNA (sc-37007) or human GCN2 siRNA (sc-45644) or human ATF4 siRNA (sc-35112) or human MetRS siRNA (sc-75775) (Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Immunofluorescence, Staining, Northern Blot, Two Tailed Test, Transfection, Control, Western Blot

Journal: Biomedicines

Article Title: Ferulic Acid Alleviates Chemotherapy-Induced POI by Targeting the Grp78 and Perk-eIF2α-ATF4-CHOP Pathway to Attenuate Endoplasmic Reticulum Stress

doi: 10.3390/biomedicines14030714

Figure Lengend Snippet: Effect of FA on Grp78 expression and on the Perk/eIF2α/ATF4/CHOP pathway in POI mice. ( A ) Representative immunohistochemical images of Grp78 of each group, scale bar = 100 μm. ( B ) Representative images of Grp78 protein bands in the ovaries of each group. ( C ) Statistical analysis of Grp78 protein expression in each group ( n = 5–6). ( D ) Relative expression of Grp78 mRNA in ovaries of each group ( n = 6). ( E ) Representative images of ovarian p-Perk, Perk, p-eIF2α, eIF2α, ATF4, and CHOP protein bands in each group. ( F – I ) Statistical analysis of p-Perk/Perk, p-eIF2α/eIF2α, ATF4, and CHOP expression in each group ( n = 5–8). ( J – M ) Relative expression of Perk , eIF2α , Atf4 , and Chop mRNA in the ovaries of each group ( n = 6). ( N ) Representative images of ovarian ERO1α protein bands in each group. ( O ) Statistical analysis of ERO1α expression in each group ( n = 3). # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. model group.

Article Snippet: Antibodies used in this experiment include: β-ACTIN (Proteintech, Wuhan, China, 20536-1-AP, 66009-1-Ig), AMH (Proteintech, 14461-1-AP), ATF4 (Proteintech, 60035-1-Ig), BAX (Proteintech, 60267-1-Ig), BCL-2 (Abcam, Cambridge, UK, ab182858), BCL-xL (Proteintech, 10783-1-AP), BMP15 (Proteintech, 18982-1-AP), Caspase 3 (Proteintech, 19677-1-AP), CHOP (Proteintech, 66741-1-Ig), eIF2α (Proteintech, 68479-1-Ig), GDF9 (Proteintech, 29309-1-AP), Grp78 (Proteintech, 11587-1-AP), p-eIF2α (CST, Danvers, MA, USA, 3398), p-Perk (Proteintech, 82534-1-RR), Perk (Proteintech, 68482-1-Ig), Biotin Conjugated AffiniPure Goat Anti-Rabbit IgG(H + L) (Boster, Wuhan, China, BA1003), DyLight 594 Conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (Boster, BA1142), Anti-rabbit IgG (H + L) (DyLight 800 4X PEG Conjugate) (CST, 5151), Anti-mouse IgG (H + L) (DyLight 680 Conjugate) (CST, 5470).

Techniques: Expressing, Immunohistochemical staining, Control

Journal: Biomedicines

Article Title: Ferulic Acid Alleviates Chemotherapy-Induced POI by Targeting the Grp78 and Perk-eIF2α-ATF4-CHOP Pathway to Attenuate Endoplasmic Reticulum Stress

doi: 10.3390/biomedicines14030714

Figure Lengend Snippet: Effect of FA on Grp78 and the Perk/eIF2α/ATF4/CHOP pathway in 4-OHCP-damaged KGN cells. ( A ) Relative expression of Grp78 , Perk , eIF2α , Atf4 , and Chop mRNA in cells of each group ( n = 5–7). ( B ) Representative immunofluorescence images of Grp78 of each group, scale bar = 50 μm. ( C ) Representative images of Grp78 protein bands of each group. ( D ) Statistical analysis of Grp78 protein expression of each group ( n = 5). ( E ) Representative images of p-Perk/Perk, p-eIF2α/eIF2α, ATF4, and CHOP protein bands in the indicated groups. ( F – I ) Statistical analysis of the protein levels shown in ( E ) ( n = 5–8). ( J ) Representative images of Grp78, p-Perk/Perk, p-eIF2α/eIF2α, ATF4, and CHOP protein bands in KGN cells treated with or without TM. ( K – O ) Statistical analysis of the protein levels shown in ( J ) ( n = 3–5). # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. model group; † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. FA group.

Article Snippet: Antibodies used in this experiment include: β-ACTIN (Proteintech, Wuhan, China, 20536-1-AP, 66009-1-Ig), AMH (Proteintech, 14461-1-AP), ATF4 (Proteintech, 60035-1-Ig), BAX (Proteintech, 60267-1-Ig), BCL-2 (Abcam, Cambridge, UK, ab182858), BCL-xL (Proteintech, 10783-1-AP), BMP15 (Proteintech, 18982-1-AP), Caspase 3 (Proteintech, 19677-1-AP), CHOP (Proteintech, 66741-1-Ig), eIF2α (Proteintech, 68479-1-Ig), GDF9 (Proteintech, 29309-1-AP), Grp78 (Proteintech, 11587-1-AP), p-eIF2α (CST, Danvers, MA, USA, 3398), p-Perk (Proteintech, 82534-1-RR), Perk (Proteintech, 68482-1-Ig), Biotin Conjugated AffiniPure Goat Anti-Rabbit IgG(H + L) (Boster, Wuhan, China, BA1003), DyLight 594 Conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (Boster, BA1142), Anti-rabbit IgG (H + L) (DyLight 800 4X PEG Conjugate) (CST, 5151), Anti-mouse IgG (H + L) (DyLight 680 Conjugate) (CST, 5470).

Techniques: Expressing, Immunofluorescence, Control

Journal: Science translational medicine

Article Title: Pancreatitis is an FGF21-deficient state that is corrected by replacement therapy

doi: 10.1126/scitranslmed.aay5186

Figure Lengend Snippet: (A) H&E staining of pancreata from a representative normal and pancreatitis patient. (B to G) Pancreatic concentrations of FGF21 (B and C), ATF3 (D and E), and ATF4 (F and G) in normal individuals and patients with chronic (CP) and acute (AP) pancreatitis. Representative immunofluorescence images are shown in (B), (D), and (F), and quantification of samples (normal, n = 14; chronic, n = 49; and acute pancreatitis, n = 3) is shown in (C), (E), and (G). In the immunofluorescence images, DAPI staining is blue and FGF21, ATF3, and ATF4 stainings are gray. Scale bar indicates 50 μm for all images. Results in (C), (E), and (G) are expressed as means ± SEM. Statistical differences (P > 0.05) are indicated between all values that have different letters. R.F.U., relative fluorescence units.

Article Snippet: Mouse ATF3 and ATF4 expression plasmids were purchased from GenScript.

Techniques: Staining, Immunofluorescence, Fluorescence

Journal: Science translational medicine

Article Title: Pancreatitis is an FGF21-deficient state that is corrected by replacement therapy

doi: 10.1126/scitranslmed.aay5186

Figure Lengend Snippet: (A) Immunoblot of pancreatic ATF4 and ATF3 at 4, 8, 12, or 18 hours after the first cerulein injection in CIP with β-actin as loading control. (B and C) Quantification of ATF4 (B) and ATF3 (C) in (A). (D) Schematic of the mouse −1497/+5 bp region of the Fgf21 promoter with the sequences and positions of the wild-type (WT) and mutant amino acid response elements (AARE). (E to H) ChIP assays quantified by qPCR for ATF4 (E and G) and ATF3 (F and H) binding to the AARE1 + 3 or AARE2 response elements from mouse pancreas in the CIP time-course experiment. IgG, immunoglobulin G. Results are expressed as means ± SEM. n = 3 to 4 mice per group for all experiments. *P < 0.05; **P < 0.01; ****P < 0.0001. Asterisks indicate statistically different values relative to vehicle.

Article Snippet: Mouse ATF3 and ATF4 expression plasmids were purchased from GenScript.

Techniques: Western Blot, Injection, Control, Mutagenesis, Binding Assay

Journal: Science translational medicine

Article Title: Pancreatitis is an FGF21-deficient state that is corrected by replacement therapy

doi: 10.1126/scitranslmed.aay5186

Figure Lengend Snippet: (A) Fgf21 mRNA in 266–6 mouse acinar cells treated with vehicle (black bar) or 100 nM cerulein (white bar) for 12 hours (N = 3). (B and C) Repression of the Fgf21 promoter using a luciferase reporter gene evaluated after Atf3 siRNA knockdown (N = 4) (B) or ATF3 overexpression (N = 4) (C) in 266–6 cells treated as in (A). (D) Wild-type and AARE mutant Fgf21 promoter luciferase (Luc) activity in 266–6 cells overexpressing ATF3 and ATF4 (N = 4). (E) Fgf21 promoter luciferase activity in 266–6 cells expressing increasing amounts (0.01 to 100 ng) of ATF3 expression plasmid and a fixed amount (1 ng) of ATF4 expression plasmid (N = 8). (F) Truncated Fgf21 promoter luciferase assays in 266–6 cells overexpressing ATF3 and ATF4 (N = 6). (G) Wild-type and AARE mutant Fgf21 promoter luciferase activity in 266–6 cells treated as in (A) (N = 4). Results are expressed as means ± SEM. N, number of replicates. ***P < 0.001; ****P < 0.0001. Asterisks indicate statistically different values relative to vehicle. Statistical differences (P > 0.05) are indicated between all values that have different letters.

Article Snippet: Mouse ATF3 and ATF4 expression plasmids were purchased from GenScript.

Techniques: Luciferase, Knockdown, Over Expression, Mutagenesis, Activity Assay, Expressing, Plasmid Preparation