Journal: International Journal of Molecular Sciences

Article Title: Strontium Attenuates Hippocampal Damage via Suppressing Neuroinflammation in High-Fat Diet-Induced NAFLD Mice

doi: 10.3390/ijms241210248

Figure Lengend Snippet: Sr restrained the HFD-induced apoptosis by altering expression levels of proteins related to the ERS pathway. ( A ) Western blot analysis of caspase-3, GRP78, IRE1α, p-IRE1α, XBP1, eIF2α, p-eIF2α, ATF4, ATF6, CHOP, and β-actin. ( B – K ) Relative protein expression of caspase-3 ( B ), GRP78 ( C ), IRE1α ( D ), p-IRE1α ( E ), XBP1 ( F ), eIF2α ( G ), p-eIF2α ( H ), ATF4 ( I ), ATF6 ( J ), and CHOP ( K ) in the hippocampi of each group of mice was examined through Western blotting ( n = 6 per group). Data were normalized with respect to the band of β-actin: the expression of target protein = the intensity of target protein band/the intensity of β-actin band. Results are shown as the ratio of the experimental group to the control group, and the values of the control group were taken as 1. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Subsequently, the following primary antibodies were used to incubate the membranes overnight at 4 °C: rabbit anti- NF-κB (#8242), rabbit anti- p38 (#9212), rabbit anti- ERK (#9102), rabbit anti-phospho- ERK ( p-ERK , #4370), rabbit anti-phospho- p38 ( p-p38 , #4511), and anti- caspase-3 (#9662) (purchased from Cell Signaling Technology (Danvers, MA, USA)); mouse anti- ATF6 (EM1701-94) (purchased from Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China); rabbit anti- XBP1 (A1731), rabbit anti-phospho- NF-κB ( p- NF-κB , AP0475), rabbit anti- GRP78 (A0241), and mouse anti- β-actin (purchased from Wuhan ABclonal Technology Co., Ltd., Wuhan, China); rabbit anti- eIF2α (ab115822), rabbit anti- TLR4 (ab13556), and rabbit anti-phospho- eIF2α ( p-eIF2α , ab32157) (purchased from Abcam (Cambridge, MA, USA)); and rabbit anti- CHOP (BM4962), anti-phospho- IRE1α ( p-IRE1α , BM4444), rabbit anti- ATF4 (BM5179), and rabbit anti- IRE1α (A00683-1) (purchased from Wuhan BOSTER Biological Technology Co., Ltd., Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Repeated Exposure to D -Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

doi: 10.3389/fnmol.2016.00165

Figure Lengend Snippet: Sequences of PCR primers.

Article Snippet: Primary antibodies against p-eIF2α (Ser51) (1:1000; Cell Signaling, #3398), eIF2α (1:1000; Cell Signaling, #5324), p-eEF2 (Thr56) (1:1000; Cell Signaling, #2331), p-p70S6K (Thr389) (1:1000; Cell Signaling, #9234), p-4EBP1 (Thr37/46) (1:500; Cell Signaling, #2855), 4EBP1 (1:500; Cell Signaling, #9644), OPHN1 (1:1000; Cell Signaling, #11939), ATF4 (1:1000; NeuroMab, #75-345), MAP2 (1:2000; Sigma, #M4403) from Sigma, CaMKIIa (1:1000; Millipore, #05-532), puromycin [1:1000; ( )], and β-actin (1:40000; Abcam, #AB6276) were used.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Repeated Exposure to D -Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

doi: 10.3389/fnmol.2016.00165

Figure Lengend Snippet: Repeated d-amphetamine administration increases the translation of a subset of uORF-containing mRNAs. (A,B,E,G) Relative mRNA expression levels of Ophn1 (A) , Atf4 (B) , Ppp1r15a (E) , and Ddit3 (G) in non-polysomal (NP) and polysomal (P) fractions analyzed by qRT-PCR in the striatum of mice chronically treated with D -amphetamine (10 mg/kg, once daily for 5 days) or saline ( n = 5 mice/group). All candidate mRNAs were normalized to β- actin or Gapdh mRNA and expressed as a percentage of saline control. (C,D) Representative western blot (top) and quantification (bottom) of OPHN1 (C) and ATF4 (D) (normalized to β-actin) expression levels in the striatum 60 or 120 min after the last injection of saline (sal) or D -amphetamine ( D -amph). Data are expressed as a percentage of saline control ( n = 5 mice/group). (F,H) Ratio of non-polysomal (NP) and polysomal (P) fractions of Ppp1r15a (F) and Ddit3 (H) mRNAs from the results represented in (E,G) , respectively. Results are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01 by unpaired Student t -test (saline versus D -amphetamine).

Article Snippet: Primary antibodies against p-eIF2α (Ser51) (1:1000; Cell Signaling, #3398), eIF2α (1:1000; Cell Signaling, #5324), p-eEF2 (Thr56) (1:1000; Cell Signaling, #2331), p-p70S6K (Thr389) (1:1000; Cell Signaling, #9234), p-4EBP1 (Thr37/46) (1:500; Cell Signaling, #2855), 4EBP1 (1:500; Cell Signaling, #9644), OPHN1 (1:1000; Cell Signaling, #11939), ATF4 (1:1000; NeuroMab, #75-345), MAP2 (1:2000; Sigma, #M4403) from Sigma, CaMKIIa (1:1000; Millipore, #05-532), puromycin [1:1000; ( )], and β-actin (1:40000; Abcam, #AB6276) were used.

Techniques: Expressing, Quantitative RT-PCR, Saline, Control, Western Blot, Injection

Journal: Cell Reports Medicine

Article Title: Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis

doi: 10.1016/j.xcrm.2024.101728

Figure Lengend Snippet: ATF4 is regulated by NAT10 through ac4C modification (A) Volcano plot showing the mRNA expression of NAT10-KO compared to control cells. Red dots indicate upregulated genes (fold change >1.25, adjusted p value <0.05), blue dots indicate downregulated genes (fold change <0.75, adjusted p value <0.05). (B) Gene profiling showing the ac4C modification distribution of NAT10-KO cells compared to that of control cells. (C) Volcano plot showing the ac4C peak of NAT10-KO compared to control cells. Red dots indicate upregulated peaks (fold change >1.25, adjusted p value <0.05), while blue dots indicate downregulated peaks (fold change <0.75, adjusted p value <0.05). (D) Sequence logo of representative motifs within ac4C peaks. (E) Venn diagram showing the intersection between downregulated genes (fold change <0.75, adjusted p value <0.05) and peaks (fold change <0.75, adjusted p value <0.05) of NAT10-KO cells compared to control cells for the 143B and HOS cell lines. (F) Correlation analysis of NAT10 expression with each of the overlapping 9 genes from (E) in the RNA-seq analysis of osteosarcoma patient samples. The red dot represents ATF4. (G) Views of ac4C modification peaks of ATF4 in the 143B cell line from acRIP-seq. (H) GSEA of ATF4 targets in the 143B cell line, by permutation test. (I) RT-qPCR analysis of ATF4 mRNA from RIP by ac4C antibody (143B [left] and HOS [right] cell lines) ( n = 3). (J) RT-qPCR analysis of ATF4 mRNA in 143B (left) and HOS (right) cells ( n = 3). (K) ATF4 protein levels in NAT10-KO cell lines measured by immunoblotting (143B [left] and HOS [right]). (L) Changes in ATF4 mRNA stability measured by RT-qPCR in the indicated groups. Decay graphs were generated by applying the one-phase decay model. Extra sum-of-squares F test. Half-life time calculated using a linear model ( n = 3). (M) Diagram depicting the workflow of detection of ac4C site in ATF4 transcript using chemical reduction method. (N) Sanger sequence detected the ac4C site in ATF4 transcript (C > T misincorporation). (O) Misincorporation rates of ATF4 transcript in control and NAT10-KO cell ( n = 3). (P) Dual-luciferase reporter assays of wild-type or mutated ac4C sites ATF4 sequence in NAT10-KO 143B (left) and HOS (right) cells ( n = 3). (Q) Sequence of ATF4 in WT and ac4C-MUT group detected by Sanger sequencing. (R) RIP-qPCR analysis of ATF4 mRNA using ac4C antibody in WT and ac4C-MUT group ( n = 3). (S) ATF4 mRNA stability measured by RT-qPCR in WT and ac4C-MUT group. Decay graphs were generated by applying the one-phase decay model. Extra sum-of-squares F test. Half-life time calculated using a linear model ( n = 3). (T–V) Proliferation (T), colony formation (U), and migration and invasion (V) of WT and ac4C-MUT group, Scale bar: 100 μm ( n = 3). Data are presented as the mean ± SD; ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, by one-way ANOVA with Dunnett’s multiple comparisons test (I, J, and P), with Tukey’s multiple comparisons test (O and R), by two-way ANOVA with Sidak’s multiple comparisons test (T), and by Student’s t test (U).

Article Snippet: ATF4 Antibody , Boster Bio , Cat# A00371-4.

Techniques: Modification, Expressing, Control, Sequencing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Generated, Luciferase, Migration

Journal: Cell Reports Medicine

Article Title: Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis

doi: 10.1016/j.xcrm.2024.101728

Figure Lengend Snippet: NAT10 regulates ASNS expression and Asn biosynthesis via ATF4 transcriptional regulation (A) RNA-seq showed that ATF4 target ASNS was downregulated in NAT10-KO 143B (left) and HOS (right) cell lines. (B) ASNS is the enzyme responsible for Asn biosynthesis. (C) ASNS expression in NAT10-KO cell lines by immunoblotting. (D) Asn level in NAT10-KO 143B (top) and HOS (bottom) cells by UHPLC-MS/MS targeted amino acids ( n = 3). (E and F) Asn (E) and Asp (F) levels in NAT10-KO 143B (top) and HOS (bottom) cells measured by indicated kit ( n = 3). (G) qPCR analysis of ASNS promoter signal from ChIP (using ATF4 antibody) ( n = 3). (H) Blot of ASNS promoter ChIP signal (using ATF4 antibody) by PCR and DNA gel electrophoresis. (I) Schematic diagram of the dual-luciferase plasmid. (J) Dual-luciferase reporter assays of ASNS promoter activity in ATF4-KD 143B (left) and HOS (right) cells ( n = 3). (K) Protein level of ASNS in ATF4-KD 143B (left) and HOS (right) cells. (L) Asn levels in ATF4-KD 143B (left) and HOS (right) cells measured by ELISA ( n = 3). (M) Dual-luciferase reporter assays of ASNS promoter activity of ATF4-OE in NAT10-KO 143B (left) and HOS (right) cells ( n = 3). (N) ASNS protein level of ATF4-OE in NAT10-KO 143B (left) and HOS (right) cells. (O) Asn level of ATF4-OE in NAT10-KO 143B (left) and HOS (right) cells measured by ELISA ( n = 3). (P–R) Proliferation (P), colony formation (Q), and migration (R) of ATF4-KD 143B and HOS cells. Scale bar: 100 μm ( n = 3). (S) ASNS protein levels in ASNS-KO 143B (left) and HOS (right) cells. (T) Diagram depicting the workflow of flux assay across using N 15 -labeled Asp. (U) N 15 -labeled Asn amount in ASNS-KO and control cell lines ( n = 3). Data are presented as the mean ± SD; ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, by Student’s t test (G), by one-way ANOVA with Dunnett’s multiple comparisons test (E, F, L, Q, and U), with Tukey’s multiple comparisons test (J, M, and O), and two-way ANOVA with Dunnett’s multiple comparisons test (P).

Article Snippet: ATF4 Antibody , Boster Bio , Cat# A00371-4.

Techniques: Expressing, RNA Sequencing, Western Blot, Tandem Mass Spectroscopy, DNA Gel Electrophoresis, Luciferase, Plasmid Preparation, Activity Assay, Enzyme-linked Immunosorbent Assay, Migration, Flux Assay, Labeling, Control

Journal: Cell Reports Medicine

Article Title: Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis

doi: 10.1016/j.xcrm.2024.101728

Figure Lengend Snippet: NAT10 promotes osteosarcoma progression via ATF4/ASNS/Asn in vivo and in vitro (A) Overexpression of ATF4 and ASNS in NAT10-KO 143B (left) and HOS (right) cell lines. (B) Proliferation assay of ATF4-OE, ASNS-OE, and Asn (0.1 mM)-treated NAT10-KO 143B (left) and HOS (right) cells ( n = 3). (C–E) Colony formation (C), migration (D), and invasion (E) of ATF4-OE, ASNS-OE, and Asn (0.1 mM)-treated NAT10-KO 143B (left) and HOS (right) cells. Scale bar: 100 μm ( n = 3). (F) Global protein synthesis of ATF4-OE, ASNS-OE, and Asn (0.1 mM)-treated NAT10-KO 143B (left) and HOS (right) cells. (G and H) Tumor growth (G) and survival (H) of the ATF4-OE, ASNS-OE, and Asn-treated (0.25 mmol/kg every two days by intraperitoneal injection [i.p.]) mouse models ( n = 8 per group), by log-rank test in (H). (I) Quantification of lung metastasis in the mouse model ( n = 8 per group). (J) Representative H&E images showing lung metastasis nodules of the mouse model. Scale bars: 500 μm (left), 200 μm (right). (K) Representative IHC images showing the expression of NAT10, ATF4, ASNS, and Ki-67 in the mouse model. Scale bars: 50 μm (left), 25 μm (right). (L) Kaplan-Meier analysis showing overall survival and LMFS curves generated for patients stratified according to the protein levels of NAT10, ATF4, and ASNS, by log-rank test. (M) ROC analysis of three marker combinations (NAT10, ATF4, and ASNS) and NAT10 in OS (left) (combination: AUC = 0.772, NAT10: AUC = 0.645) and LMFS (right) (combination: AUC = 0.817, NAT10: AUC = 0.688) in the osteosarcoma patient cohort, by Venkatraman method test. Data are presented as the mean ± SD; ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, by one-way ANOVA with Tukey’s multiple comparisons test (C and I) and two-way ANOVA with Tukey’s multiple comparisons test (B).

Article Snippet: ATF4 Antibody , Boster Bio , Cat# A00371-4.

Techniques: In Vivo, In Vitro, Over Expression, Proliferation Assay, Migration, Injection, Expressing, Generated, Marker

Journal: Cell Reports Medicine

Article Title: Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis

doi: 10.1016/j.xcrm.2024.101728

Figure Lengend Snippet: Structure-based virtual screen identified paliperidone and AG-401 as potential inhibitors of NAT10 (A) Diagram depicting the workflow of screening potential inhibitors of NAT10. (B) Docking score of molecular docking in NAT10 inhibitor screening for FDA-approved drugs and the Specs compound library. (C) mRNA ac4C modification level of 143B cells treated with the top 20 ranked compounds (20 μM, 24 h) from molecular docking by dot blot. (D) ITC assay between NAT10 and paliperidone (left) or NAT10 and AG-401 (right). (E) mRNA ac4C modification level of 143B (left) and HOS (right) cells treated with paliperidone, AG-401, and Remodelin at the indicated concentrations (24 h) by dot blot. (F–I) Proliferation (F), colony formation (G and H), and migration (I) of 143B (left) and HOS (right) cells treated with paliperidone or AG-401 at the indicated concentrations ( n = 3). (J) NAT10, ATF4, and ASNS protein levels in 143B (left) and HOS (right) cells treated with paliperidone or AG-401 (24 h) at the indicated concentrations. (K) Asn levels in 143B (left) and HOS (right) cells treated with paliperidone or AG-401 (24 h) at the indicated concentrations ( n = 3). (L and M) Tumor growth (L) and survival (M) of the mouse model treated with paliperidone (2 mg/kg daily, i.p.) and AG-401 (5 mg/kg daily, i.p.), by log-rank test in (M) ( n = 8 per group). (N) Quantification of mouse lung metastasis in the mouse model ( n = 8 per group). (O) Representative H&E images showing lung metastasis nodules of the mouse model. Scale bars: 500 μm (left), 200 μm (right). (P) Representative IHC images showing the expression of NAT10, ATF4, ASNS, and Ki-67 in the mouse model. Scale bars: 50 μm (left), 25 μm (right). Data are presented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, by one-way ANOVA with Dunnett’s multiple comparisons test (K and N), with Tukey’s multiple comparisons test (G and H), and two-way ANOVA with Dunnett’s multiple comparisons test (F).

Article Snippet: ATF4 Antibody , Boster Bio , Cat# A00371-4.

Techniques: Drug discovery, Modification, Dot Blot, Isothermal Titration Calorimetry, Migration, Expressing

Journal: Cell Reports Medicine

Article Title: Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis

doi: 10.1016/j.xcrm.2024.101728

Figure Lengend Snippet: Combination of paliperidone and AG-401 inhibited osteosarcoma progression in organoid and PDX models (A) Diagram depicting the workflow of drug synergy assay and establishment of PDX models and organoids. (B and C) Cell viability (B) and Bliss score (C) for paliperidone and AG-401 combined treatment. (D) Cell viability of organoids derived from patient 1 (P1) treated with paliperidone (8 μM), AG-401 (22 μM), or combination (paliperidone: 5 μM, AG-401: 10 μM) ( n = 3). (E) Representative images showing organoids derived from patient and treated with paliperidone, AG-401, or combination. Scale bars: 200 μm. (F) Quantification of area of organoids derived from patient 1 (P1) treated with paliperidone, AG-401, or combination.. (G and H) Tumor growth (G) and survival (H) of the PDX model treated with paliperidone (2 mg/kg daily, i.p.), AG-401 (5 mg/kg daily, i.p.), or combination (paliperidone: 1 mg/kg daily, i.p., AG-401: 2.5 mg/kg daily, i.p.) ( n = 5 per group). (I) Body weight of the PDX model treated with paliperidone, AG-401, or combination ( n = 5 per group). (J) Representative IHC images showing the expression of NAT10, ATF4, ASNS, and Ki-67 in the PDX model. Scale bars: 50 μm (left), 25 μm (right) ( n = 5 per group). Data are presented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, by one-way ANOVA with Dunnett’s multiple comparisons test (F and J), two-way ANOVA with Dunnett’s multiple comparisons test (D), and log-rank tests (H).

Article Snippet: ATF4 Antibody , Boster Bio , Cat# A00371-4.

Techniques: Derivative Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis

doi: 10.1016/j.xcrm.2024.101728

Figure Lengend Snippet:

Article Snippet: ATF4 Antibody , Boster Bio , Cat# A00371-4.

Techniques: Virus, Recombinant, Transfection, Bicinchoninic Acid Protein Assay, CCK-8 Assay, Staining, Immunohistochemistry, Membrane, Invasion Assay, Luciferase, Reporter Assay, Cell Viability Assay, Sequencing, Software