Journal: Oncotarget

Article Title: Discovery of selective inhibitors of Glutaminase-2, which inhibit mTORC1, activate autophagy and inhibit proliferation in cancer cells.

doi: 10.18632/oncotarget.2173

Figure Lengend Snippet: Figure 4: AV-1 induced cell autophagy through activated AMPK and ULK1. (A) Effects of AV-1 treatment on the induction of apoptosis and autophagy in HepG2 cells manifested by cleavage caspase 3 or LC3B. (B) The effects of AV-1 on AMPK activation. C. The effects of AV-1 on activation of ULK1 and BCL2 associated Beclin1. HepG2 cells were treated with vehicle (DMSO) or AV-1 for various times as indicated prior to cell lysis for western blot analysis with the antibodies indicated. The results shown are representative of three independent experiments.

Article Snippet: Antibodies to GAPDH, caspase 3, LC3B, p-ULK1(S317), p-ULK1(S757), Beclin1, p-BCL2(S70), p70S6K, p-p70S6K(T389), 4E-BP1, α-tubulin, 4E-BP, p-4EBP(S65), p-eIF4B(S422), mTOR, p-mTOR(S2481), p-mTOR(S2448), p-Raptor(S792), Tuberin, p-Tuberin(T1462), p-Tuberin(T1387), p-AMPK(T172) and p-AMPK(S108) were from Cell Signaling Technology; antibody to p-Beclin1(S15) was from Abbiotec; antibodies to KGA and Raptor were from Abcam; antibodies to BCL2 were from Upstate Biotechnology; antibodies to α-tubulin were from Chemicon International; antibodies to ULK1, β-tublin and AMPK were from GeneTex; antibodies to GAB were from abnova and Gene Tex; antibodies to GAC were from ProteinTech Group; antibodies to p-mTOR(S2446) and AMPK were from Millipore; anti-mouse and anti-rabbit IgGs were obtained from PerkinElmer.

Techniques: Activation Assay, Lysis, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Silencing TUFM Inhibits Development of Monocrotaline-Induced Pulmonary Hypertension by Regulating Mitochondrial Autophagy via AMPK/mTOR Signal Pathway

doi: 10.1155/2022/4931611

Figure Lengend Snippet: Alleviated mitophagy by TUFM silence is associated with AMPK/mTOR pathway. (a) Representative western blot bands for TUFM, AMPK, p-AMPK, mTOR, p-mTOR, BECN1, Atg13, Atg16L1, ULK1, and Atg12. (b–k) Densitometry data represent the intensity of each group. The data is presented as the mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The related primary antibody included TUFM (Abcam, ab173300, 1 : 10000 dilution), LC3 (CST, #3868, 1 : 1000 dilution), BECN1 (CST, #3495, 1 : 1000 dilution), P62 (CST, #39749, 1 : 1000 dilution), ATG13 (Proteintech, #18258-1-AP, 1 : 1000 dilution), α -SMA (BOSTER, BM0002, 1 : 1000 dilution), CD31 (Abcam, ab222783, 1 : 2000 dilution), Bax (Abcam, ab182734, 1 : 1000 dilution), Bcl2 (HUABIO, ET1702-53, 1 : 1000 dilution), AMPK (Bioss, bs-5551R, 1 : 1000 dilution), p-AMPK (Bioss, bs-2771R, 1 : 1000 dilution), mTOR (Bioss, bs-3494R, 1 : 1000 dilution), p-mTOR (Bioss, bs-3495R, 1 : 1000 dilution), ULK1 (BOSTER, A00584-1, 1 : 1000 dilution), Apaf-1 (HUABIO, ET1607-12, 1 : 1000 dilution), ATG12 (CST, #4180, 1 : 1000 dilution), ATG16L1 (CST, #8089, 1 : 1000 dilution), and β -actin (CST, #4970, 1 : 1000 dilution) which was used as an internal reference.

Techniques: Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Silencing TUFM Inhibits Development of Monocrotaline-Induced Pulmonary Hypertension by Regulating Mitochondrial Autophagy via AMPK/mTOR Signal Pathway

doi: 10.1155/2022/4931611

Figure Lengend Snippet: Schematic figure of the current study. Increased TUFM expression in hypoxia-stimulated pulmonary arterial hypertension cells causes an increasing mtDNA translation, leading to dysfunction of the mitochondrial respiratory chain. Mitochondrial dysfunction induces cellular stress and then activates AMPK. On the one hand, activated AMPK decreases the phosphorylation level of mTOR, inhibits the activity of mTOR, and then disassociates from ULK1. Thus, phosphorylation of specific sites of ULK1 and Atg13 is released. Meanwhile, the ULK1 complex is activated through autophosphorylation at thr180 and phosphorylates Atg13, FIP200, atg101, and other Atg proteins. The activated ULK1 complex then translocates to the isolation membrane of the endoplasmic reticulum, where autophagy is initiated. On the other hand, activated AMPK will directly stimulate ULK1 and BECN1, initiating autophagy.

Article Snippet: The related primary antibody included TUFM (Abcam, ab173300, 1 : 10000 dilution), LC3 (CST, #3868, 1 : 1000 dilution), BECN1 (CST, #3495, 1 : 1000 dilution), P62 (CST, #39749, 1 : 1000 dilution), ATG13 (Proteintech, #18258-1-AP, 1 : 1000 dilution), α -SMA (BOSTER, BM0002, 1 : 1000 dilution), CD31 (Abcam, ab222783, 1 : 2000 dilution), Bax (Abcam, ab182734, 1 : 1000 dilution), Bcl2 (HUABIO, ET1702-53, 1 : 1000 dilution), AMPK (Bioss, bs-5551R, 1 : 1000 dilution), p-AMPK (Bioss, bs-2771R, 1 : 1000 dilution), mTOR (Bioss, bs-3494R, 1 : 1000 dilution), p-mTOR (Bioss, bs-3495R, 1 : 1000 dilution), ULK1 (BOSTER, A00584-1, 1 : 1000 dilution), Apaf-1 (HUABIO, ET1607-12, 1 : 1000 dilution), ATG12 (CST, #4180, 1 : 1000 dilution), ATG16L1 (CST, #8089, 1 : 1000 dilution), and β -actin (CST, #4970, 1 : 1000 dilution) which was used as an internal reference.

Techniques: Expressing, Phospho-proteomics, Activity Assay, Isolation, Membrane