Journal: Biological Research

Article Title: The CB1 cannabinoid receptor regulates autophagy in the tibialis anterior skeletal muscle in mice

doi: 10.1186/s40659-023-00426-5

Figure Lengend Snippet: Knockout CB1 prevents LC3 II accumulation in tibialis anterior muscle. p62/Sqstm1, LC3, AKT 473 , total AKT, mTOR 2448 , total mTOR, p70S6K 389 , total p70S6K, AMPK 172 , and total AMPK protein levels were determined by western blot. A Representative western blot image. B p62/Sqstm1 protein levels. C LC3 I protein levels. D LC3 II normalized by total LC3. E Representative western blot image. F AKT 473 . G mTOR 2448 . H p70S6K 389 . I AMPK 172 . Two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. Statistical significance was set at p < 0.05. Values are expressed as mean and S.E.M. and scatter dot plots, as appropriate

Article Snippet: The primary antibodies used were CB1 (Cayman #10006590), LC3A/B (CST #4108), p62/Sqstm1 (NBP1-48320, Novus), GAPDH (CST #5174), phospho-p70S6K Thr389 (CST #9234), p70S6K Total (CST #2708), phospho-AMPKα Thr172 (CST #2535), AMPKα Total (CST #5831), phospho-Akt Ser473 (CST #9271), Akt Total (CST #9272), phospho-mTOR Ser2448 (CST #2971), mTOR total (CST #2972), Lamp1 (CST #3243), Lamp2 (CST #49,067), and K48-linkage Specific Polyubiquitin (CST # 4289).

Techniques: Knock-Out, Western Blot

Journal: bioRxiv

Article Title: NRF2 connects Src tyrosine kinase to ferroptosis resistance in glioblastoma

doi: 10.1101/2023.05.08.539792

Figure Lengend Snippet: Immunoblotting of p-Src Y416 and Src in U87-MG, T98G and GBMSC83 cells treated with DAS 10nM (U87-MG and T98G) and 1μM (GBMSC83) (left) or PP2 5μM (right) for 24hrs. Vinculin was used as loading control. S2 . Immunoblotting of p-Src Y416 and Src in U87-MG cells treated with DAS 10nM or Trigonelline 5μM for 16hrs. S3 . Immunoblotting and relative densitometric analysis of NRF2 targets, HO-1 and NQO1, in U87-MG and T98G cells after 24hrs of transient transfections with active Src (Src Y527F ) or catalytically inactive mutant (Src K295M ). S4 . Immunoblotting of p-p70S6K T389 and p70S6K in U87-MG and T98G cells treated with Rapamycin 100nM for 48hrs. Actin was used as loading control. S5 . Immunofluorescence staining in U87-MG cells treated with Rapamycin 100nM for 48hrs. NRF2 (green); DNA (Hoechst, blu). S6 . Confocal microscopy analyses of U87-MG cells treated with Rapamycin 100nM for 48hrs. p62 (green); KEAP1 (red); DNA (Hoechst, blue); 4x digital magnification showing merged signals. S7 . Volcano plot. The log2 FC indicates the mean activity level for each pathway. Each dot represents one pathway. Black and blue dots represent pathways with no significant p-value and no significant FC respectively, red dots represent up-and down-regulated pathways in GBM (TCGA data) compared with normal brain cortex (GTEx data); S10 . Rapresentative Bright Field images and propidium iodide (PI + , red) staining in T98G cells after 48hrs irradiation (10Gy), with or without DAS 10nM and/or Erastin (ERA) 1μM. Statistical analyses: paired ( S3 ) and unpaired ( S9,S10 ) Student’s t-test: (* p < 0.05; ** p < 0.01).

Article Snippet: Primary antibodies used are as follows: anti-NRF2 (D1Z9C) (Cell Signaling Technology), anti-p62 (SQSTM1) (PM045, MBL International), anti-phospho-p62 (SQSTM1) (Ser349) (PM074, MBL International), anti-Vinculin (E1E9V) (Cell Signaling Technology), anti-Lamin (E-1) (sc-376248, Santa Cruz Biotechnology), anti-GAPDH (D16H11) (Cell Signaling Technology); anti-Src (2108S) (Cell Signaling Technology), anti-phospho-Src (Tyr416) (2101S) (Cell Signaling Technology), anti-KEAP1 (G-2) (sc-365626, Santa Cruz Biotechnology), anti-Heme Oxygenase 1 (A-3) (sc-136960, Santa Cruz Biotechnology), anti-NQO1 (A-180) (sc-32793, Santa Cruz Biotechnology), anti-p70S6K (Cell Signaling Technology), anti-phospho-p70S6K (Thr389) (Cell Signaling Technology), anti-ERK 1/2 (137F5) (Cell Signaling Technology), anti-phospho-ERK 1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology), anti-Ubiquitin (P4D1, Santa Cruz Biotechnology).

Techniques: Western Blot, Transfection, Mutagenesis, Immunofluorescence, Staining, Confocal Microscopy, Activity Assay, Irradiation

Journal: bioRxiv

Article Title: NRF2 connects Src tyrosine kinase to ferroptosis resistance in glioblastoma

doi: 10.1101/2023.05.08.539792

Figure Lengend Snippet: A ) Immunoblotting of p-p70S6K T389 and p70S6K in U87-MG, T98G and GBMSC83 cells treated for 16 hrs with DAS 10nM and 1μM, respectively. B ) Immunoblotting of NRF2, p-p62 S349 , p62, HO-1 and NQO1 in U87-MG and T98G cells upon Rapamycin 100nM treatment for 48hrs, and relative NRF2 densitometric analysis.

Article Snippet: Primary antibodies used are as follows: anti-NRF2 (D1Z9C) (Cell Signaling Technology), anti-p62 (SQSTM1) (PM045, MBL International), anti-phospho-p62 (SQSTM1) (Ser349) (PM074, MBL International), anti-Vinculin (E1E9V) (Cell Signaling Technology), anti-Lamin (E-1) (sc-376248, Santa Cruz Biotechnology), anti-GAPDH (D16H11) (Cell Signaling Technology); anti-Src (2108S) (Cell Signaling Technology), anti-phospho-Src (Tyr416) (2101S) (Cell Signaling Technology), anti-KEAP1 (G-2) (sc-365626, Santa Cruz Biotechnology), anti-Heme Oxygenase 1 (A-3) (sc-136960, Santa Cruz Biotechnology), anti-NQO1 (A-180) (sc-32793, Santa Cruz Biotechnology), anti-p70S6K (Cell Signaling Technology), anti-phospho-p70S6K (Thr389) (Cell Signaling Technology), anti-ERK 1/2 (137F5) (Cell Signaling Technology), anti-phospho-ERK 1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology), anti-Ubiquitin (P4D1, Santa Cruz Biotechnology).

Techniques: Western Blot

Journal: Frontiers in Endocrinology

Article Title: Insulin, dibutyryl-cAMP, and glucose modulate expression of patatin-like domain containing protein 7 in cultured human myotubes

doi: 10.3389/fendo.2023.1139303

Figure Lengend Snippet: List of primary antibodies used for immunoblotting.

Article Snippet: p-p70S6K Thr 389 , #9205 , Rabbit , 1:1000 , Cell Signaling Technology Inc, USA.

Techniques: Western Blot

Journal: Frontiers in Endocrinology

Article Title: Insulin, dibutyryl-cAMP, and glucose modulate expression of patatin-like domain containing protein 7 in cultured human myotubes

doi: 10.3389/fendo.2023.1139303

Figure Lengend Snippet: Functional assessment of PNPLA7 in cultured human myoblasts. Myoblasts were treated with siRNA against PNPLA7 mRNA (siPNPLA7) or scrambled siRNA (siSCR). Functional consequences of PNPLA7 knock-down (A–K) were estimated by measuring (A) the abundance of α1-subunit of Na + ,K + -ATPase (NKAα1), (B) phosphorylation of p70S6K Thr389 , (C) phosphorylation of S6RP Ser235/236 , (D) phosphorylation of 4E-BP1 Thr 37/46 , (E) the abundance of ACC, (F) ACC1 mRNA (gene ACACA ), (G) ACC2 mRNA (gene ACACB ), (H) phosphorylation of ACC Ser79 , (I) phosphorylation of AMPK Thr172 , (J) IL6 mRNA, and (K) phosphorylation of STAT3 Tyr705 . Ponceau S staining was used as a control of loading and transfer of immunoblotting (A–E, H–K) . (L) A representative Ponceau-stained membrane. Actin blots, which were used as an additional loading control are shown in the corresponding Supplementary Figure . The mRNA levels of IL-6, ACC1, and ACC2, which were measured using qPCR, are presented as geometric mean of expression ratios against two reference genes, 18S rRNA and ACTB. Results are means with SEM (n = 7). *p < 0.05 vs. siSCR.

Article Snippet: p-p70S6K Thr 389 , #9205 , Rabbit , 1:1000 , Cell Signaling Technology Inc, USA.

Techniques: Functional Assay, Cell Culture, Staining, Western Blot, Expressing

Journal: Journal of Cell Science

Article Title: The ecl family gene ecl3 + is induced by phosphate starvation and contributes to sexual differentiation in fission yeast

doi: 10.1242/jcs.260759

Figure Lengend Snippet: Some phenotypes of Δ ecls cells were not restored by TORC1 suppression. (A) JS183 (WT) and JS184 (Δ ecls ) cells were grown in EMM to OD 600 =0.5 and then transferred into phosphate-depleted EMM. Phosphorylation levels of Psk1 at 0, 3 and 6 h after phosphate starvation (−P) were examined using western blotting (left). Quantitative results of phosphorylation levels (P-Psk1/γ-tubulin) are shown in the right panel ( n =3). (B) WT (FY7288) and Δ ecls (FY7288Δ ecl1/2/3 ) were grown in EMM to OD 600 =0.5 (control) and then transferred into phosphate-depleted (−P) EMM (24 h) with and without 200 ng ml −1 rapamycin and 9 mM caffeine (RC). Rum1–HA levels were measured using western blotting (left). Quantitative results are shown in the right panel ( n =3). (C) Left panel: JY808 (WT) and JY808Δ ecl1/2/3 (Δ ecls ) cells were grown in EMM to OD 600 =0.5 and then transferred into phosphate-depleted EMM with and without 200 ng ml −1 rapamycin and 9 mM caffeine (RC). Each mating rate was measured ( n =3). Right panel: the diploid cells (JY333×HM3802) (WT) and JY333Δ ecl1/2/3 ×HM3802Δ ecl1/2/3 (Δ ecls ) were grown in EMM to OD 600 =0.5 and then transferred into phosphate-depleted EMM with and without 200 ng ml −1 rapamycin and 9 mM caffeine (RC). Each sporulation rate was measured ( n =3). (D) JY333 (WT) and JY333Δ ecl1/2/3 (Δ ecls ) cells were grown in EMM to OD 600 =0.5 and then transferred into phosphate-depleted (−P) EMM with and without 200 ng ml −1 rapamycin and 9 mM caffeine. DNA content was measured by flow cytometry. All graphs show PI intensities from 0 to 50,000. Data show the mean±s.d.

Article Snippet: Rum1–HA was detected with the anti-HA (12CA5) antibody (1:10,000; Roche, 11666606001), Cdc2 with the anti-CDK1 antibody (Y100.4) (1:10,000; Abcam, ab5467), Psk1–myc with the MYC/c-Myc antibody (1:2000; Santa Cruz Biotechnology, sc-40), phospho-Psk1-myc with the anti-phospho-Psk1/RPS6KB1/p70 S6 kinase (Thr389) antibody (1:1000; Cell Signaling Technology, 9206), γ-tubulin with the monoclonal anti-Gtb1/γ-tubulin antibody (1:2000; Sigma-Aldrich, T5326), and α-tubulin with the monoclonal anti-α-tubulin antibody (1:10,000; Sigma-Aldrich, T6074).

Techniques: Western Blot, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of mitochondrial phosphate carrier prevents high phosphate-induced superoxide generation and vascular calcification

doi: 10.1038/s12276-023-00950-0

Figure Lengend Snippet: a Mitochondrial superoxide production in control and butyl malonate (BMA)-treated primary vascular smooth muscle cells (pVSMCs) was determined using mitoSOX (# experiments; n = 9). b Immunoblotting of ERK1/2 and p70S6K was performed (# experiments; n = 3). c The transcript levels of osteogenic genes were estimated using quantitative PCR (# experiments; n = 3). d Cell viability was evaluated using an MTT assay (# experiments; n = 5). e Calcification was quantitated using Alizarin Red S staining (# experiments; n = 3). f Calcification of rat aortic rings after high-Pi incubation ex vivo with and without BMA treatment for 7 days as visualized by Alizarin Red S or von Kossa staining. g The calcium content in rat aortic rings was measured using a Quantichrom Calcium Assay Kit (# rats; n = 5). The data were analyzed using one-way ANOVA. * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: After blocking, the membranes were washed three times with 0.1% TBST for 10 min each and incubated with primary antibodies against the following proteins overnight at 4 °C: β-Actin (Abcam, Cambridge, MA, USA, #ab6276), p-ERK1/2 (Cell Signaling, Danvers, MA, USA, #4370P), ERK1/2 (Cell Signaling, #9102), p-p70S6K (Cell Signaling, #9205S), and p70S6K (Cell Signaling, #9202).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, MTT Assay, Staining, Incubation, Ex Vivo, Calcium Assay