Journal: bioRxiv

Article Title: Autophagy is Regulated by Mitochondrial Calcium Transporters NCLX and MCU

doi: 10.1101/2023.03.17.533187

Figure Lengend Snippet: Western blot analysis of AML12 cells 72 hours after transfection with NCLX (siNCLX), MCU (siMCU), or negative control (siNC) siRNAs and quantification of ( A ) total ULK1 protein levels, ( B ) ULK1 phosphorylation at Serine 757 (S757), ( C ) p-ULK1 (S757) relative to total ULK1, ( D ) total 4E-BP1 protein levels, ( E ) 4E-BP1 phosphorylation at Threonine 37/46, ( F ) p-4E-BP1 relative to total 4E-BP1, ( G ) ULK1 phosphorylation at Serine 555 (S555) and ( H ) p-ULK1 (S555) relative to total ULK1 (n = 5). ( I ) Oxygen consumption rates (OCR) linked to ATP synthesis (oligomycin-sensitive respiration) of transfected AML12 cells in complete media (CM) (n = 4). Data were analyzed by one-way RM ANOVA followed by Bonferroni’s multiple comparison test. Bar graphs show mean ± SD, dots in each group represent biological replicates and dots of equal color indicate paired replicates. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant.

Article Snippet: Primary antibodies against LC3 A/B (12741; 1:1,000), LC3B (; 1:1,000), ATG7 (8558; 1:1,000), ATG12 (4180; 1:1,000), ULK1 (8054; 1:500), p-ULK S555 (5869, 1:500), p-ULK S757 (14202; 1:500), 4E-BP1 (9644; 1:1,000), p-4E-BP1 (2855; 1:1,000), MCU (14997; 1:1,000) and α-tubulin (3873; 1:10,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Negative Control

Journal: bioRxiv

Article Title: Autophagy is Regulated by Mitochondrial Calcium Transporters NCLX and MCU

doi: 10.1101/2023.03.17.533187

Figure Lengend Snippet: ( A ) Measurements of intramitochondrial Ca 2+ levels to evaluate efflux rates after 100 µM ATP stimulation in the presence of vehicle (DMSO) or 10 µM CGP 37157 (CGP) (n = 3). ( B) Cytosolic Ca 2+ measurements using Fura-2 AM after stimulation with 100 µM ATP; bar graphs showing quantifications of Ca 2+ peaks and the half-life of one phase decay after the peak (n = 4). Data were analyzed by two-tailed paired t test. ( C) Western blot analysis for LC3 II and ( D ) LC3 I after 1 hour exposure to either complete media (CM) or HBSS in the presence of either DMSO or 10 µM CGP (n = 6). ( E ) Western blot analysis of LC3 II after 1 hour exposure to HBSS in the absence or presence of 100 nM Bafilomycin A1 during the last 30 min of treatment (n = 6). Data were analyzed by two-way randomized blocks ANOVA followed by Tukey’s multiple comparisons test. ( F ) LC3 II net flux of DMSO- and CGP-treated cells after 1 hour HBSS exposure. Data were analyzed by two-tailed unpaired t test. ( G ) Western blots for LC3 II and (H) LC3 I after 3 hours exposure to complete media (CM) in the presence of vehicle (DMSO) or 200 nM rapamycin and in the presence of either DMSO or 10 µM CGP (n = 6). ( I ) Western blot analysis of LC3 II after 3 hours of 200 nM rapamycin treatment in the absence or presence of 100 nM Bafilomycin A1 during the last hour of treatment (n = 6). Data were analyzed by two-way randomized blocks ANOVA followed by Tukey’s multiple comparisons test. ( J ) LC3 II net flux in DMSO- and CGP-treated cells in the presence of 200 nM rapamycin. Data were analyzed by two-tailed unpaired t test. ( K ) Western blot analysis of phosphorylated 4E-BP1 in DMSO or CGP-treated cells after HBSS (n = 5) or ( L ) rapamycin (n = 3) stimulation. ( M ) Western blots for LC3 I and LC3 II in AML12 cells pre-incubated with 50 µM BAPTA-AM for 1 hour (n = 6). Data were analyzed by two-way randomized blocks ANOVA followed by Tukey’s multiple comparisons test. Data are presented in columns with means or means ± SD. Dots in each group represent biological replicates and paired biological replicates are represented by equal color dots. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

Article Snippet: Primary antibodies against LC3 A/B (12741; 1:1,000), LC3B (; 1:1,000), ATG7 (8558; 1:1,000), ATG12 (4180; 1:1,000), ULK1 (8054; 1:500), p-ULK S555 (5869, 1:500), p-ULK S757 (14202; 1:500), 4E-BP1 (9644; 1:1,000), p-4E-BP1 (2855; 1:1,000), MCU (14997; 1:1,000) and α-tubulin (3873; 1:10,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Two Tailed Test, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Immunogenic cell death triggered by impaired deubiquitination in multiple myeloma relies on dysregulated type I interferon signaling

doi: 10.3389/fimmu.2023.982720

Figure Lengend Snippet: Western-blot analysis of the ubiquitin, ISR and UPR expression profiles in NCI-H929 cells exposed to BTZ, ONX-0914, RA190 or PR619. (A) NCI-H929 exposed to PR619 (1,5 µM) or left untreated were subjected to protein extraction and subsequent SDS-PAGE/western blotting using antibodies specific for ubiquitin and actin (loading control), as indicated. Shown is one representative experiment out of three. (B) Equal amounts of protein lysates derived from NCI-H929 exposed to a 12-h treatment with DMSO, BTZ (50 nM), ONX-0914 (50 nM), RA190 (50 nM) or PR619 (1,5 µM) were analyzed by SDS-PAGE/western-blotting using antibodies directed against PKR, (p)PKR, GCN2, (p)GCN2, eIF2α, (p)eIF2α, 4E-BP1, (p)4E-BP1 and tubulin (loading control), as indicated. Shown is one representative experiment out of three. (C) NCI-H929 whole cell-lysates described in (B) were further assessed for their contents in PERK, (p)PERK, IRE1, (p)IRE1, ATF6 by SDS-PAGE/western-blotting, as indicated. Equal protein loading was ensured by probing the membranes with an actin antibody. Shown is one representative experiment out of three. (D) Densitometric analysis of phosphorylated of PERK, IRE1, PKR and GCN2 normalized to total proteins and reported as foldchange relative to DMSO.

Article Snippet: After a 20-min incubation with 1X Roti ® -Block (Carl Roth ® ) at room temperature, membranes were incubated overnight at 4°C with primary antibodies specific for β1 (clone MCP421), β2 (clone MCP165), α6 (clone MCP20), ubiquitin (clone FK2) all purchased from Enzo Life Sciences, Inc. Other primary antibodies include anti-TCF11 (clone D5B10), anti-PERK (clone C33E10), anti-(p)PERK (#3179), anti-IRE1α (#3294), anti-ATF6 (clone D4Z8V), anti-PKR (1297), anti-GCN2 (65981), anti-eIF2α (9722), anti-(p)eiF2α (9721), anti-4E-BP1 (clone 53H11), anti-(p)4E-BP1 (2855s), anti-GAPDH (clone 14C10), anti-caspase-3 (9662S), anti-cleaved caspase-3 (9661L), anti-TBK1 (3013), anti-(p)TBK1 (clone D52C2), anti-IRF3 (4302), anti-STAT1 (clone 2x) and anti-(p)STAT1 (clone 58D6) all products from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Protein Extraction, SDS Page, Derivative Assay

Journal: Breast Cancer : Targets and Therapy

Article Title: Dual Target of EGFR and mTOR Suppresses Triple-Negative Breast Cancer Cell Growth by Regulating the Phosphorylation of mTOR Downstream Proteins

doi: 10.2147/BCTT.S390017

Figure Lengend Snippet: Western blot analysis of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) pathways in the MDA-MB-468 cell line treated with gefitinib and everolimus and their relative expression. ( A ) Western blotting of the expression patterns of the EGFR and mTOR pathway proteins after treatment with gefitinib and everolimus alone and in combination ( P < 0.05). ( B – I ) Expression of EGFR, phosphorylated EGFR, mTOR, phosphorylated mTOR, ribosomal protein S6 kinase (S6K1), phosphorylated S6K1, 4E binding protein 1 (4EBP1), and phosphorylated 4EBP1 proteins after intervention with gefitinib and everolimus alone and in combination ( P < 0.05).

Article Snippet: Primary rabbit mAb to mTOR (2972), p-mTOR (Ser2448) (2971), 4EBP1 (9452), p-4EBP1 (Thr37/46) (2855), p-70S6 Kinase (9202), p-70S6 Kinase (9205), EGFR (2232), and p-EGFR (D7A5) XP ® (3777) were purchased from Cell Signaling (USA).

Techniques: Western Blot, Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Anti-Aging Effects of Anthocyanin Extracts of Sambucus canadensis Caused by Targeting Mitochondrial-Induced Oxidative Stress

doi: 10.3390/ijms24021528

Figure Lengend Snippet: Anthocyanin inhibited the PI3K/Akt/mTOR signaling pathway of senescent cells. ( A , B ) Cells were treated with 40 mM D-galactose alone or in combination with 40 μg/mL anthocyanin for 72 h, and the expression of p-AKT/AKT, p-4EBP1/4EBP1 were measured by western blot. Data are presented as mean ± SD, n = 3. ** p < 0.01.

Article Snippet: Antibodies against AKT, p-AKT, 4EBP1, p-4EBP1, p70s6k, p-p70s6k were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Anti-Aging Effects of Anthocyanin Extracts of Sambucus canadensis Caused by Targeting Mitochondrial-Induced Oxidative Stress

doi: 10.3390/ijms24021528

Figure Lengend Snippet: Anthocyanin decreased the activity of PI3K/AKT/mTOR and increased apoptosis in mice lenses induced by D-galactose. ( A , B ) The expression of p-AKT/AKT, p-4EBP1/4EBP1 were measured using western blot. ( C , D ) The expression of Bcl-2 family proteins and Cleaved-caspase-3 were measured using western blot. Data are presented as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01.

Article Snippet: Antibodies against AKT, p-AKT, 4EBP1, p-4EBP1, p70s6k, p-p70s6k were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Activity Assay, Expressing, Western Blot