Journal: Frontiers in Pharmacology

Article Title: Simultaneous suppression of PKM2 and PHGDH elicits synergistic anti-cancer effect in NSCLC

doi: 10.3389/fphar.2023.1200538

Figure Lengend Snippet: PKM2-IN-1 in combination with NCT-503 induces cell cycle arrest via the AMPK-mediated signaling pathway, and inhibits GLUT1 expression. (A) Cell cycle distributions were measured by flow cytometry after treatment with PKM2-IN-1, NCT-503, and a combination of the two agents. (B) Statistical analysis of the cell cycle distribution. (C) The levels of G2/M phase arrest-related proteins were measured by Western blotting after treatment with PKM2-IN-1, NCT-503, and combination of the two agents. (D) Intracellular ATP level of A549 cells was examined after 24 h treatment with combination of PKM2-IN-1 and NCT-503. (E) The levels of p-AMPK, p-mTOR, p-p70S6K, p-53, and GLUT1 were determined by Western blotting. * p < 0.05 and ** p < 0.01.

Article Snippet: Antibodies against cyclin B1, cdc2, caspase 3, poly (ADP-ribose) polymerase (PARP), Bax, B-cell lymphoma 2 protein (Bcl-2), pyruvate kinase (PK) M2, phospho-AMP-activated protein kinase (AMPK)α (Thr172), phospho-mTOR (Ser2448), phospho-p70 ribosomal protein S6 Kinase (p70S6K) (Thr389), phospho-histone H2AX (Ser139) (γ-H2AX), p-ATM (Ser 1981), β-actin, and HRP-conjugated goat anti-rabbit and horse anti-mouse secondary antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, United States).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells

doi: 10.3390/ijms24109075

Figure Lengend Snippet: Torin2-mediated mTOR inhibition counteracted TMZ+AT101/AT101-regulated chemoresistance mechanisms of surviving GBM cells. Primary GBM cells were co-cultured with microglia and astrocytes in defined cellular proportions mimicking an incomplete GBM resection. The cultures were treated with TMZ+AT101/AT101 (50 µM TMZ, 2.5 µM AT101), Torin2 (0.5 nM), or a combination of Torin2 and TMZ+AT101/AT101 for six days. Western blotting analysis on p-mTOR, p-P70S6K, and p-4E-BP1 of co-cultured primary cultures a (PCa, A ) was performed after stimulation for six days, respectively. The obtained signals were normalized to glycerinaldehyde 3-phosphate dehydrogenase (GAPDH) used as loading control. Exemplary data shown; n = 2 biological replicates. Death rates of PCa were obtained by performing a cytotoxicity assay after three and six days of stimulation, respectively ( n = 2 biological replicates with n = 2 technical replicates) ( B ). Gene expression of different pro-tumorigenic markers in PCa after three or six days of treatment was determined by qRT-PCR ( C ) ( n = 2 biological replicates with n = 2 technical replicates). The induction of gene expression upon stimulation is displayed as n-fold expression changes relative to the DMSO controls after normalization to GAPDH. The significances between different time points were determined by a two-way ANOVA test followed by Tukey’s multiple comparison test and are indicated on a line linking the bars. Significant differences compared to the DMSO control were determined by a non-paired t -test and are indicated directly above the bars (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Used primary antibodies were anti-phospho-GSK3b (1:250, rabbit; #9336, Cell Signaling, Danvers, MA, USA), anti-phospho-Akt (1:250, rabbit; #4060, Cell Signaling), anti-phospho-mTOR (1:250, rabbit; #2971, Cell Signaling), anti-phospho-4E-BP1 (1:1000, rabbit; #2855, Cell Signaling), and anti-phospho-P70S6K (1:1000, rabbit; #9205, Cell Signaling) in 5% bovine serum albumin/tris-buffered saline with 0.1% Tween 20 (TBST).

Techniques: Inhibition, Cell Culture, Western Blot, Cytotoxicity Assay, Expressing, Quantitative RT-PCR

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: iScience

Article Title: TIAR and FMRP shape pro-survival nascent proteome of leukemia cells in the bone marrow microenvironment

doi: 10.1016/j.isci.2023.106543

Figure Lengend Snippet:

Article Snippet: Antibodies used to develop the Western blots were: anti-ATF4 (Proteintech, #10835-I-AP), anti-α-tubulin (Calbiochem, #CP06-100UG), anti-β-actin (Sigma Aldrich, #A5316), anti-eIF2α (CellSignaling, #2103), anti-phospho Ser51-eIF2α (Cell Signaling, #9721), anti-eIF4E (Cell Signaling, #2067), anti-phospho Ser209-eIF4E (Cell Signaling, #9741), anti-FMRP (Bethyl, #A305-199A), anti-FXR1P (Santa Cruz Biotechnology, #sc-374148), anti-FXR2P (Santa Cruz Biotechnology, #sc-376963), anti-GAPDH (Bethyl, #A300-639A), anti-G3BP1 (Proteintech, #66486-1-Ig), anti-HIF1α (Cell Signaling, #14179), anti-HIF2α (Bethyl, #A700-003-T), anti-HuR (Santa Cruz Biotechnology, #sc-5261), anti-PABP (Proteintech, #10970-1-AP), anti-PARP1 (CellSignaling, #9532), anti-PPP5C (Proteintech, #11715-1-AP), anti-phospho Ser371-p70S6K (CellSignaling, #9208), anti-RPL6 (Proteintech, #15387-1-AP), anti-RPL10 (Cell Signaling, #72912), anti-RPL17 (Proteintech, #67223-1-Ig), anti-RPL18 (Proteintech, #17029-1-AP), anti-RPL21 (Proteintech, #15226-1-AP), anti-RPL28 (Proteintech, #16649-1-AP), anti-RPL31 (Proteintech, #16497-1-AP), anti-RPL37A (Proteintech, #14660-1-AP), anti-RPS2 (Abcam, #ab264336), anti-RPS3 (Proteintech, #66046-1-Ig), anti-RPS6 (Proteintech, #66886-1-Ig), anti-RPS15 (Proteintech, #14957-1-AP), anti-Tia-1 (Proteintech, #12133-2-AP), anti-TIAR (Bethyl, #A303-613A), anti-mTOR (CellSignaling, #2983), anti-phospho Ser2448–mTOR (CellSingaling, #5536), anti-Smad3 (Proteitech, #66516-1-Ig), anti-phospho Ser423/425-Smad3 (CellSignaling, #9520), anti-ZW10 (Proteintech, #24561-1-AP), and secondary antibodies: conjugated with horseradish peroxidase (HRP) goat anti-mouse (Dako, #P0447) and goat anti-rabbit (Dako, #P0448) or StarBright Blue 700 conjugated goat anti-rabbit IgG (Bio-Rad, #12004162) and StarBright Blue 520 conjugated goat anti-mouse IgG (Bio-Rad, #12005867).

Techniques: Purification, shRNA, Transduction, Recombinant, Reporter Assay, Lysis, Plasmid Preparation, Software

Journal: iScience

Article Title: TIAR and FMRP shape pro-survival nascent proteome of leukemia cells in the bone marrow microenvironment

doi: 10.1016/j.isci.2023.106543

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho Ser371-p70S6K , CellSignaling , Cat #9208.

Techniques: Purification, shRNA, Transduction, Recombinant, Reporter Assay, Lysis, Plasmid Preparation, Software

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: 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