sirna against mouse ddit4 sc-142310  (Santa Cruz Biotechnology)

Journal: Biomedical Reports

Article Title: Resveratrol ameliorates high‑fat diet‑induced insulin resistance via the DDIT4/mTOR pathway in skeletal muscle

doi: 10.3892/br.2025.1977

Figure Lengend Snippet: Expression of DDIT4, mTOR, GLUT4, PI3K, and AKT in MHY1485-treated IR C2C12 cells. (A) Protein expression levels of DDIT4, mTOR, GLUT4, PI3K, AKT after the activation of mTOR. (B) Semi-quantification of p-protein/t-protein ratios for mTOR, PI3K, AKT and protein expression levels of DDIT4, GLUT4 after MHY1485 treatment. (C) Glucose levels in culture medium after MHY1485 treatment. (D) Cell TG levels after MHY1485 treatment. Data are presented as the mean ± SD (n=3). * P<0.05 vs. the CON group; # P<0.05 vs. the PA group; & P<0.05 vs. the PA + RSV group. DDIT4, DNA-damage-inducible transcript 4; mTOR, mammalian target of rapamycin; GLUT4, glucose transporter 4; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; IR, insulin resistance; p-, phosphorylated; t-, total; TG, triglyceride; CON, control; PA, palmitic acid; RSV, resveratrol.

Article Snippet: Some of the PA + RSV group cells were again divided into two groups: The PA + RSV + MHY1485 group C2C12 cells were treated with 10 μM MHY1485 (cat. no. HY-B0795; MedChemExpress) to activate mTOR, and the PA + RSV + DDIT4-siRNA group C2C12 cells were transfected with a small interfering (si)RNA against DDIT4 (DDIT4-siRNA; Shanghai Genechem Co. Ltd.), all incubated at 37 ̊C for 24 h. The cells were collected for western blotting and cellular TG content determination.

Techniques: Expressing, Activation Assay, Control

Journal: Biomedical Reports

Article Title: Resveratrol ameliorates high‑fat diet‑induced insulin resistance via the DDIT4/mTOR pathway in skeletal muscle

doi: 10.3892/br.2025.1977

Figure Lengend Snippet: Expression levels of DDIT4, mTOR, GLUT4, PI3K, and AKT in DDIT4-siRNA-treated IR C2C12 cells. (A) DDIT4-siRNA-1 had the most significant silencing effect. (B) Protein expression levels of DDIT4, mTOR, GLUT4, PI3K, and AKT after DDIT4-siRNA treatment. (C) Semi-quantification of p-protein/t-protein ratios for mTOR, PI3K, and AKT and protein expression levels of DDIT4 and GLUT4 after DDIT4-siRNA treatment. (D) Glucose levels in culture medium after DDIT4-siRNA treatment. (E) TG levels of C2C12 cells after DDIT4-siRNA treatment. Data are presented as the mean ± SD (n=3). * P<0.05 vs. the CON group; # P<0.05 vs. the PA group; & P<0.05 vs. the PA + RSV group. DDIT4, DNA-damage-inducible transcript 4; mTOR, mammalian target of rapamycin; GLUT4, glucose transporter 4; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; siRNA, small interfering RNA; IR, insulin resistance; p-, phosphorylated; t-, total; TG, triglyceride; CON, control; PA, palmitic acid; RSV, resveratrol.

Article Snippet: Some of the PA + RSV group cells were again divided into two groups: The PA + RSV + MHY1485 group C2C12 cells were treated with 10 μM MHY1485 (cat. no. HY-B0795; MedChemExpress) to activate mTOR, and the PA + RSV + DDIT4-siRNA group C2C12 cells were transfected with a small interfering (si)RNA against DDIT4 (DDIT4-siRNA; Shanghai Genechem Co. Ltd.), all incubated at 37 ̊C for 24 h. The cells were collected for western blotting and cellular TG content determination.

Techniques: Expressing, Small Interfering RNA, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: The PERK-eIF2α-ATF4 Axis Is Involved in Mediating ER-Stress-Induced Ferroptosis via DDIT4-mTORC1 Inhibition and Acetaminophen-Induced Hepatotoxicity.

doi: 10.3390/antiox14030307

Figure Lengend Snippet: Figure 4. ATF4-induced DDIT4 and subsequent mTORC1 inhibition trigger ferroptotic signaling. (A) Western blot analysis of indicated proteins in AML12 cells after Torin1 treatment (0, 0.5, 1, and 2 µM) for 24 h. (B–D) AML12 cells were treated with erastin (5 µM) or Torin1 (0.5 µM) in the presence or absence of Fer-1 (10 µM) for 16 h. (B) Lipid ROS was stained with BODIPY™581/591 C11 and measured by flow cytometry. (C) Cell viability assessed by MTT. (D) Quantification of mRNA level of Ptgs2. (E) Western blot analysis of indicated proteins in AML12 after treatment with erastin (10 µM) at indicated times. (F–I) For knockdown Ddit4, AML12 cells were transfected with control siRNA (scRNA) or siDdit4 for 36 h. (F) Western blot analysis of indicated proteins after erastin (10 µM) treatment for 24 h. (G) Quantification of mRNA level of indicated genes. (H) Lipid ROS was stained with BODIPY™581/591 C11 and measured by flow cytometry. (I) Cell viability assessed by MTT assay after erastin treatment (0, 1.25, 2.5, 5, 10, and 20 µM) for 24 h. (J) Atf4−/−cells were infected with Ad-GFP or Ad-ATF4 and then treated with erastin (10 µM) for 24 h. Western blot analysis of indicated proteins. (K–L) Ad-GFP and Ad-ATF4 were infected in scRNA or siDdit4-transfected AML12 cells followed by erastin (10 µM) treatment for 24 h. (K) Western blot analysis of indicated proteins. (L) Lipid ROS was stained with BODIPY™581/591 C11 and measured by flow cytometry. Data are mean ± SD (n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001, and NS, not significant.

Article Snippet: Small Interfering RNA Transfection Small interfering RNA (siRNA) against mouse Ddit4 (siDdit4) (sc-142310, Santa Cruz, Dallas, TX, USA) was purchased from Santa Cruz Biotechnology, and negative control siRNA (scRNA) (AM4611, Ambion, Austin, TX, USA) was purchased from Ambion.

Techniques: Inhibition, Western Blot, Staining, Flow Cytometry, Knockdown, Transfection, Control, MTT Assay, Infection

Journal: Antioxidants

Article Title: The PERK-eIF2α-ATF4 Axis Is Involved in Mediating ER-Stress-Induced Ferroptosis via DDIT4-mTORC1 Inhibition and Acetaminophen-Induced Hepatotoxicity

doi: 10.3390/antiox14030307

Figure Lengend Snippet: List of primers for qRT-PCR analysis of gene expression. F—forward, R—reverse, bs—bases.

Article Snippet: Small interfering RNA (siRNA) against mouse Ddit4 ( siDdit4 ) (sc-142310, Santa Cruz, Dallas, TX, USA) was purchased from Santa Cruz Biotechnology, and negative control siRNA ( scRNA ) (AM4611, Ambion, Austin, TX, USA) was purchased from Ambion.

Techniques: Gene Expression, Sequencing

Journal: Antioxidants

Article Title: The PERK-eIF2α-ATF4 Axis Is Involved in Mediating ER-Stress-Induced Ferroptosis via DDIT4-mTORC1 Inhibition and Acetaminophen-Induced Hepatotoxicity

doi: 10.3390/antiox14030307

Figure Lengend Snippet: ATF4-induced DDIT4 and subsequent mTORC1 inhibition trigger ferroptotic signaling. ( A ) Western blot analysis of indicated proteins in AML12 cells after Torin1 treatment (0, 0.5, 1, and 2 μM) for 24 h. ( B – D ) AML12 cells were treated with erastin (5 μM) or Torin1 (0.5 μM) in the presence or absence of Fer-1 (10 μM) for 16 h. ( B ) Lipid ROS was stained with BODIPY™ 581/591 C11 and measured by flow cytometry. ( C ) Cell viability assessed by MTT. ( D ) Quantification of mRNA level of Ptgs2 . ( E ) Western blot analysis of indicated proteins in AML12 after treatment with erastin (10 μM) at indicated times. ( F – I ) For knockdown Ddit4 , AML12 cells were transfected with control siRNA ( scRNA ) or siDdit4 for 36 h. ( F ) Western blot analysis of indicated proteins after erastin (10 μM) treatment for 24 h. ( G ) Quantification of mRNA level of indicated genes. ( H ) Lipid ROS was stained with BODIPY™ 581/591 C11 and measured by flow cytometry. ( I ) Cell viability assessed by MTT assay after erastin treatment (0, 1.25, 2.5, 5, 10, and 20 μM) for 24 h. ( J ) Atf4 −/− cells were infected with Ad-GFP or Ad-ATF4 and then treated with erastin (10 μM) for 24 h. Western blot analysis of indicated proteins. ( K – L ) Ad-GFP and Ad-ATF4 were infected in scRNA or siDdit4 -transfected AML12 cells followed by erastin (10 μM) treatment for 24 h. ( K ) Western blot analysis of indicated proteins. ( L ) Lipid ROS was stained with BODIPY™ 581/591 C11 and measured by flow cytometry. Data are mean ± SD ( n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001, and NS, not significant.

Article Snippet: Small interfering RNA (siRNA) against mouse Ddit4 ( siDdit4 ) (sc-142310, Santa Cruz, Dallas, TX, USA) was purchased from Santa Cruz Biotechnology, and negative control siRNA ( scRNA ) (AM4611, Ambion, Austin, TX, USA) was purchased from Ambion.

Techniques: Inhibition, Western Blot, Staining, Flow Cytometry, Knockdown, Transfection, Control, MTT Assay, Infection