Journal: Nutrients

Article Title: Vitamin C Activates Osteoblastogenesis and Inhibits Osteoclastogenesis via Wnt/β-Catenin/ATF4 Signaling Pathways

doi: 10.3390/nu11030506

Figure Lengend Snippet: Expression of both osteoblast- and osteoclast-regulated proteins in vitamin C-treated rat tibias. ( A ) Western blot image of Wnt3a, β-catenin, and ATF4 and quantitative assay of Wnt3a, β-catenin, and ATF4 protein expression in vitamin C-treated rat tibias. ( B ) Western blot image of p-AKT, p-ERK, p-p38, and p-JNK and quantitative assay of p-AKT, p-ERK, p-p38, and p-JNK protein expression in vitamin C-treated rat tibias. Expression was quantified using ImageJ software relative to that of β-actin. Values represent the mean ± standard deviation. Values with different letters were significantly different according to Duncan’s multiple range test ( P < 0.05).

Article Snippet: Membranes were blocked with 5% bovine serum albumin prior to incubation with specific primary antibodies against BMP-2, RUNX2, Wnt3a, osteocalcin, COL-1 (Abcam, Cambridge, UK), SMAD1/5/8 (Santa Cruz Biotechnology, Dallas, TX, USA), ATF4 (Boster, Pleasanton, CA, USA), osteoprotegerin (OPG), RANK, RANKL (Bioss Antibodies, Woburn, MA, USA), TRAP, cathepsin K (GeneTex, Irvine, CA, USA), β-catenin, phosphorylated serine/threonine kinase (p-AKT), phosphorylated extracellular signal-regulated kinase (p-ERK), p-p38, phosphorylated c-Jun N-terminal kinase (p-JNK), and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Software, Standard Deviation

Journal: FEBS Open Bio

Article Title: Pyruvate alleviates high glucose‐induced endoplasmic reticulum stress and apoptosis in HK‐2 cells

doi: 10.1002/2211-5463.12834

Figure Lengend Snippet: Pyr effects on ER stress‐related protein and ROS level. HK‐2 cells were treated with Pyr (0.5 m m ) and/or HG (30 m m glucose) for 3 days. The protein expressions (A) of GRP78, CHOP, ATF4 and p‐EIF2α of HK‐2 cell were detected by western blot analyses. The percentages of GRP78 (B), CHOP (C), ATF4 (D) and p‐EIF2α (E)/β‐actin in the bar graphs were quantified by imagej software. Expressions of GRP78 (B), CHOP (C), ATF4 (D) and p‐EIF2α (E) were decreased after Pyr treatments, which ascertained that exogenous Pyr ameliorated the ER stress in HK‐2 cells. (F) HK‐2 cells were treated with Pyr (0.5 m m ) and/or HG (30 m m glucose) for 3 days. Further, HG‐induced ROS increases were inhibited by Pyr treatments in HK‐2 cells. Con: cells were treated with 5 m m glucose in the DMEM; HG: cells were treated with 30 m m glucose in the DMEM; HG + Pyr: cells were treated with 30 m m glucose and 0.5 m m Pyr in the DMEM; Pyr: cells were treated with 5 m m glucose and 0.5 m m Pyr in the DMEM. Values were means ± SEM ( n = 5). Data were analyzed by one‐way ANOVA. * P < 0.05; ** P < 0.01.

Article Snippet: The primary antibodies against Bcl‐2, BAX, Caspase‐3, glucose‐regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4) and phosphorylate α‐subunit of eukaryotic initiation factor 2 (p‐EIF2α) and the secondary antibodies (anti‐mouse IgG, anti‐rabbit IgG) were obtained from Proteintech Group, Inc (Chicago, IL, USA), and the antibody against p‐EIF2α was purchased from CST (Danvers, MA, USA).

Techniques: Western Blot, Software

Journal: Oncology reports

Article Title: Gonadotropins promote human ovarian cancer cell migration and invasion via a cyclooxygenase 2-dependent pathway.

doi: 10.3892/or.2017.5784

Figure Lengend Snippet: Figure 1. FSHR and LHR were expressed in SKOV3 and HO8910 cells but did not affect cell proliferation and apoptosis. Expression of FSHR and LHR was examined by immunocytochemical staining (A), RT-PCR (B), and western blotting (C). HO8910 and SKOV3 cells were treated with FSH and LH (0, 100, and 500 mIU/ml) alone or in combination, but no changes in proliferation (D and E) and apoptosis (F) were observed.

Article Snippet: Antibodies specific to FSHR (cat. PB1120; 1:200) and LHR (cat. A01120; 1:200) were obtained from Boster.

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: Activation of GCN2/ATF4 signals in amygdalar PKC-δ neurons promotes WAT browning under leucine deprivation

doi: 10.1038/s41467-020-16662-2

Figure Lengend Snippet: a Gene expression of Atf4 and Trb3 in amygdala by RT-PCR. b Daily food intake. c Fat mass by NMR. d Subcutaneous WAT (sWAT) weight. e Representative images of hematoxylin and eosin (H&E) staining of sWAT. f sWAT cell size quantified by Image J analysis of H&E images. g Gene expression of Ucp1, Pgc1a, Cidea, Dio2 , and Prdm16 in sWAT by RT-PCR. h Representative images of immunohistochemistry (IHC) of UCP1 in sWAT. i UCP1 protein in sWAT by western blotting (left) and quantified by densitometric analysis (right); A.U.: arbitrary units. Studies for a were conducted using 14- to 15-week-old male wild-type mice fed a control (Control) or leucine-deficient [(-) L] diet for 3 days; studies for ( b – i ) were conducted using 13- to 16-week-old male PKC-δ-Cre mice receiving AAVs expressing mCherry (PKC δ − DN ATF4) or DN ATF4 (PKC δ + DN ATF4) fed a Control or (-) L diet for 3 days. Data are expressed as the mean ± SEM ( n represents number of samples and are indicated above the bar graph), with individual data points. Data were analyzed by two-tailed unpaired Student’s t test for ( a ), or one-way ANOVA followed by the SNK (Student–Newman–Keuls) test for ( b – i ). Source data are provided as a Source data file.

Article Snippet: Tissue extracts were then immunoblotted with the following primary antibodies: anti-p-eIF2α, anti-EIF2α, anti-GCN2, anti-p-HSL, anti-HSL, anti-IRE1α, and anti-p-PKA substrates (1:1000, Cell Signaling Technology, MA, USA); anti-UCP1 and anti-ATF6 (1:1000, Abcam, Cambridge, UK); anti-TH (1:1000, Merck Millipore, Frankfurter, GER); anti-ATF4 and anti-TRB3 (1:500, Santa Cruz Biotechnology, CA, USA), anti-p-GCN2 (1:500, Biorbyt, Cambridge, UK); anti-CHOP, anti-ATF4, anti-PERK, and anti-XBP1s (1:1000, Proteintech, Hubei, P.R.C.); anti-p-IRE1α (1:1000, Epitomics, CA, USA); anti-p-PERK (1:1000, Signalway Antibody, MD, USA); anti-BIP and anti-β-actin (1:2000, Sigma, MO, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Immunohistochemistry, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Activation of GCN2/ATF4 signals in amygdalar PKC-δ neurons promotes WAT browning under leucine deprivation

doi: 10.1038/s41467-020-16662-2

Figure Lengend Snippet: a Norepinephrine (NE) levels in subcutaneous WAT (sWAT) measured by ELISA kit. b Tyrosine hydroxylase (TH) proteins in sWAT by western blotting (left) and quantified by densitometric analysis (right); A.U.: arbitrary units. c Gene expression of Adrb3 in sWAT by RT-PCR. d Fat mass by NMR. e sWAT weight. f Representative images of hematoxylin and eosin (H&E) staining of sWAT. g sWAT cell size quantified by Image J analysis of H&E images. h Gene expression of Ucp1, Pgc1a, Cidea, Dio2 , and Prdm16 in sWAT by RT-PCR. i Representative images of immunohistochemistry (IHC) of UCP1 in sWAT. j UCP1 protein in sWAT by western blotting (top) and quantified by densitometric analysis (bottom). k Summary Diagram: WAT browning is induced by leucine deprivation, which is sensed by the amino acid sensor GCN2 in PKC-δ neurons of the amygdala. Activated GCN2 then subsequently stimulates ATF4 expression and increases the PKC-δ neuronal activity that promotes WAT browning via increasing the activity of the sympathetic nerve. Studies for a – c were conducted using 22- to 24-week-old male PKC-δ-Cre mice receiving AAVs expressing mCherry (PKC δ − hM4Di) or hM4Di (PKC δ + hM4Di), fed a control (Control) or leucine-deficient [(-) L] diet for 3 days; studies for d – j were conducted using 16- to 18-week-old male PKC δ − hM4Di or PKC δ + hM4Di mice injected with saline (−CL316) or CL316243 (+CL316), fed a (-) L diet for 3 days. Data are expressed as the mean ± SEM ( n represents number of samples and are indicated above the bar graph), with individual data points. Data were analyzed by one-way ANOVA followed by the SNK (Student–Newman–Keuls) test. Source data are provided as a Source data file.

Article Snippet: Tissue extracts were then immunoblotted with the following primary antibodies: anti-p-eIF2α, anti-EIF2α, anti-GCN2, anti-p-HSL, anti-HSL, anti-IRE1α, and anti-p-PKA substrates (1:1000, Cell Signaling Technology, MA, USA); anti-UCP1 and anti-ATF6 (1:1000, Abcam, Cambridge, UK); anti-TH (1:1000, Merck Millipore, Frankfurter, GER); anti-ATF4 and anti-TRB3 (1:500, Santa Cruz Biotechnology, CA, USA), anti-p-GCN2 (1:500, Biorbyt, Cambridge, UK); anti-CHOP, anti-ATF4, anti-PERK, and anti-XBP1s (1:1000, Proteintech, Hubei, P.R.C.); anti-p-IRE1α (1:1000, Epitomics, CA, USA); anti-p-PERK (1:1000, Signalway Antibody, MD, USA); anti-BIP and anti-β-actin (1:2000, Sigma, MO, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Immunohistochemistry, Activity Assay, Injection