Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: The hypothetical pathway of this study. All the important molecular and pathways were indicated. Briefly, AMPK is a preferential substrate of Compound C than Akt in normal conditions. When TiO₂ NZs were added to cells, they inhibited the expression of AMPK and mTOR proteins, then with extra addition of Compound C or phenformin turned to inhibit the candidate target of Akt, and exerted contrary effects on regulating autophagy

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Expressing

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Contrasted effects of Compound C and phenformin on TiO₂ NZs–induced fetal growth impairment and placental cell energy/autophagy responses. ( A ) Representative fetal images and corresponding average fetal length and average fetal weight in the control, TiO₂ NZs, TiO₂ NZs + phenformin, and TiO₂ NZs + Compound C groups. ( B ) Immunofluorescence analysis of autophagy levels in HTR cells following exposure to 100 µg/mL TiO₂ NZs or 100 µg/mL bulk-TiO₂. ( C ) Corresponding cellular ATP levels measured after exposure to TiO₂ NZs and bulk-TiO₂. ( D - E ) Immunofluorescence analysis of autophagy levels following exposure to 100 µg/mL TiO₂ NZs or 100 µg/mL b-TiO₂, either alone or in combination with Compound C/phenformin. Cell nuclei were stained blue with DAPI, and autophagosomes were labeled red using CY3-conjugated secondary antibodies.Scale bar = 20 μm. ( F ) Uptake and location of TiO₂ NZs in HTR-8/Svneo (HTR) cells observed by TEM after cells were treated with 100 µg/mL of TiO₂ NZs. The TiO₂ NZs were indicated by red arrows. ( G ) The morphology of mitochondria observed by TEM after cells were exposed to 100 µg/mL of TiO₂ NZs. Normal or swollen mitochondria were indicated by red arrows, respectively. Data are expressed as mean ± SD. A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( A ). One-way ANOVA was used for ( C ). ** P < 0.01, *** P < 0.001 vs. control group; $ P < 0.05 TiO₂ NZs + phenformin vs. TiO₂ NZs group; # P < 0.05 TiO₂ NZs + Compound C vs. TiO₂ NZs group

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Control, Immunofluorescence, Staining, Labeling, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Autophagosome accumulation and its modulation by Compound C and phenformin in placental cells. ( A - C ) Cell autophagy levels were assessed by immunofluorescence and observed using a confocal microscope after cells received corresponding treatments. ( D , E ) Cell autophagy levels were assessed by immunofluorescence after HTR cells were treated with Compound C and phenformin in the absence of TiO₂ NZs. ( F ) Autophagy levels were examined after Compound C was added to the starvation-induced autophagy group. Cell nuclei were stained with DAPI (blue), and autophagosomes were labeled with CY3-conjugated secondary antibodies (red). Scale bar = 100 μm

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Immunofluorescence, Microscopy, Staining, Labeling

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Compound C and phenformin differentially regulate AMPK/mTOR signaling and exhibit distinct binding modes to AKT1.( A ) Western blot analysis of AMPK, p-AMPK, mTOR, and p-mTOR expression levels in cells treated with 100 µg/mL TiO₂ NZs. ( B ) Western blot analysis of AMPK and p-AMPK expression levels in cells treated with TiO₂ NZs, either alone or in combination with Compound C or phenformin. ( C , D ) Western blot analysis of AMPK and p-AMPK expression levels in cells treated with Compound C ( C ) and phenformin ( D ). GAPDH served as a loading control. Molecular weights (kDa) are indicated beside the bands.Quantitative densitometric analysis corresponding to the western blots in ( A – D ) was performed using ImageJ software. All bands were normalized to GAPDH expression, and the tests were repeated three times. ( E ) Chemical structures of ATP-competitive AKT1 inhibitors. ( F ) Chemical structures of allosteric (auto-inhibitory) AKT1 inhibitors. ( G ) Docking conformation of Compound C at the interface of the AKT1 dimer, with AKT1a and AKT1b colored red and blue, respectively. ( H ) Docking conformation of the AKT inhibitor (4EJN) in AKT1. ( I ) Docking conformation of phenformin within the AKT1 binding site. ( J ) Superimposed binding modes of Compound C and phenformin in the AKT1 dimer. ( K, L ) Molecular docking results showing the overlapping binding region (blue frame) of Compound C and phenformin, with the upper interaction site specific to Compound C. ( M ) Docking scores (kcal/mol) of phenformin and Compound C bound to AKT1, where more negative values indicate stronger binding affinity.The data are presented as mean ± SD. An unpaired two-tailed t -test was used for ( A , C and D ). A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for (B). ** P < 0.01, *** P < 0.001 vs. control group; ### P < 0.001 vs. TiO₂ NZs group

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Binding Assay, Western Blot, Expressing, Control, Software, Two Tailed Test, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Akt-targeted autophagy modulation emerges as AMPK declines in TiO₂ NZs-treated cells. ( A ) Western blot analysis of Akt and p-Akt protein levels in cells treated with 100 µg/mL TiO₂ NZs for 24 h. ( B, C ) Protein levels of Akt and p-Akt after treatment with Compound C or phenformin. ( D, E )Western blot analysis of Akt, p-Akt, mTOR, and p-mTOR in cells treated with TiO₂ NZs alone or in combination with Compound C or phenformin for 24 h. GAPDH served as a loading control. Molecular weights (kDa) are indicated beside the bands.Quantitative densitometric analysis corresponding to the western blots in ( A – E ) was performed using ImageJ software. All bands were normalized to GAPDH expression, and the tests were repeated three times. ( F ) AMPK mRNA and protein expression levels in cells transfected with AMPK siRNA or negative control. ( G ) Immunofluorescence analysis of autophagy levels in cells transfected with AMPK siRNA alone or in combination with Compound C or phenformin. ( H ) AMPK mRNA and protein expression levels in cells transfected with AMPK overexpression vector (pcDNA3.1-AMPK) or negative control vector (pcDNA3.1). All data are presented as the mean ± SD from three independent experiments. An unpaired two-tailed t-test was used for ( A - C ). A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( D , E , F , H ). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group; ## P < 0.01, ### P < 0.001 vs. TiO₂ NZs group

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Western Blot, Control, Software, Expressing, Transfection, Negative Control, Immunofluorescence, Over Expression, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Autophagy reversal via AMPK overexpression and Akt knockdown highlights dual-target mechanism. ( A , B ) Immunofluorescence analysis of autophagy levels in HTR cells exposed to the following treatments: ( A ) Control cells, TiO₂ NZs, TiO₂ NZs + AMPK overexpression vector (pcDNA3.1-AMPK), TiO₂ NZs + AMPK overexpression vector + Compound C, or TiO₂ NZs + Compound C; ( B ) Control cells, TiO₂ NZs, TiO₂ NZs + AMPK overexpression vector, TiO₂ NZs + AMPK overexpression vector + phenformin, or TiO₂ NZs + phenformin. Cell nuclei were stained blue with DAPI, and autophagosomes were labeled red using CY3-conjugated secondary antibodies. Scale bar = 100 μm. ( C ) AKT mRNA and protein levels examined by RT-PCR and western blotting after cells were transfected with the Akt siRNA and the negative control. ( D ) Immunofluorescence analysis of autophagy levels in HTR cells exposed to the following treatments: TiO₂ NZs, TiO₂ NZs + Akt siRNA, TiO₂ NZs + Akt siRNA + Compound C, TiO₂ NZs + Compound C, TiO₂ NZs + Akt siRNA + phenformin, or TiO₂ NZs + phenformin for 24 h. Cell nuclei were stained blue with DAPI, and autophagosomes were labeled red using CY3-conjugated secondary antibodies. All data are presented as the mean ± SD. A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( C ). **** P < 0.0001 vs. NC siRNA group

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Over Expression, Knockdown, Immunofluorescence, Control, Plasmid Preparation, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Negative Control, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Nanozymes subvert pharmacological conventions: insights from counteracting the placental side effects of TiO₂ nanozymes

doi: 10.1186/s12951-026-04132-8

Figure Lengend Snippet: Phenformin and Compound C rewire AMPK/mTOR pathway in HTR cells post-TiO₂ exposure. ( A, B ) Western blot analysis of Akt, AMPK, mTOR, and their phosphorylated forms after cells were treated with 100 µg/mL TiO₂ NZs, TiO₂ NZs + AMPK overexpression vector (pcDNA3.1-AMPK), or TiO₂ NZs + AMPK overexpression vector + Compound C/phenformin for 24 h. ( C ) Protein levels of AKT, p-AKT, AMPK, p-AMPK, mTOR, p-mTOR, and LC3 in the control group, TiO₂ NZs exposure group, TiO₂ NZs + phenformin and TiO₂ NZs + Compound C groups, as determined by western blotting. GAPDH served as a loading control. Molecular weights (kDa) are indicated beside the bands. Quantitative densitometric analysis corresponding to the western blots in ( A – C ) was performed using ImageJ software. All bands were normalized to GAPDH expression, and the tests were repeated three times.Data were collected from three independent experiments and presented as mean ± SD. An unpaired two-tailed t -test was used for (C left panel). A one-way ANOVA was conducted, followed by a post-hoc Tukey’s multiple comparison test for ( A , B , C right panel). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001 TiO₂ NZs + AMPK-vector + Compound C vs. TiO₂ NZs group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 TiO₂ NZs + AMPK-vector vs. TiO₂ NZs group; † P < 0.05, †† P < 0.01, ††† P < 0.001 TiO₂ NZs + AMPK-vector + Compound C vs. TiO₂ NZs + AMPK-vector group (A-B). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group; # P < 0.05, ### P < 0.001 TiO₂ NZs + Compound C vs. TiO₂ NZs group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 TiO₂ NZs + phenformin vs. TiO₂ NZs group ( C )

Article Snippet: Compound C (an AMPK inhibitor, HY-13418 A) and phenformin (an AMPK activator, HY-16397) were bought from MedChemExpress (Monmouth Junction, NJ, USA).Bafilomycin A1 (B1793) and chloroquine (C6628) were gained from Sigma-Aldrich (St. Louis, MO, USA).Primary antibodies of AMPK (ab32047), p-AMPK (ab23875), Akt (ab8805),andp-Akt (ab38449)were supplied by Abcam (Cambridge, UK).The secondary antibody produced by CY3-conjugated goat anti-rabbit (ab6939) is a product of Abcam (Cambridge, UK).Primary antibodies against mTOR (2972), p-mTOR (5536), and LC3A/B (4108) were obtained from Cell Signaling Technology (Danvers, MA, USA).And the primary antibody of GAPDH (AF1186),BCA protein assay kit (P0010S), ATP assay kit (S0026), Trizol reagent (R0016), and cDNA synthesis kit (D7168M) were obtained from Beyotime Biotechnology (Shanghai, China).

Techniques: Western Blot, Over Expression, Plasmid Preparation, Control, Software, Expressing, Two Tailed Test, Comparison

Journal: Journal of Nanobiotechnology

Article Title: AE-MXene-modified titanium alloy promotes osseointegration by regulating the AMPK-MTOR-autophagy pathway in macrophage

doi: 10.1186/s12951-026-04080-3

Figure Lengend Snippet: AE-MXene enhanced macrophage autophagy via activation of the AMPK-mTOR signaling pathway. a ) Western blot images of p-AMPK, AMPK, p-mTOR, mTOR, ULK1, P62, Beclin1, LC3I/II, and GAPDH in RAW264.7 cells in different treatment groups. b ) Quantitative analysis of p-AMPK/AMPK ratio, p-mTOR/mTOR ratio, ULK1, P62, Beclin1, and LC3-II/LC3-I ratio in RAW264.7 cells based on Western blot images ( n ≥ 3). c ) Immunofluorescence staining images of p-AMPK, p-mTOR, P62, and LC3 in RAW264.7 cells in different treatment groups. Scale bar = 20 μm. d ) Immunofluorescence staining analysis of different treatment groups ( n ≥ 3). (e) RAW264.7 cells were pretreated with dorsomorphin (5µM) and MHY1485 (10µM) as required and then stimulated with fresh medium containing LPS for 24 h. Western blot images of p-AMPK, AMPK, p-mTOR, mTOR, ULK1, LC3I/II, and GAPDH in RAW264.7 cells in different treatment groups. (f) Quantitative analysis of p-AMPK/AMPK ratio, p-mTOR/mTOR ratio, ULK1, and LC3-II/LC3-I ratio in RAW264.7 cells based on Western blot images ( n ≥ 3). * P < 0.05, ** P < 0.01

Article Snippet: Dorsomorphin dihydrochloride (Dorso) was obtained from TargetMol Chemicals Inc. (USA).

Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: INS-1E cells were cultured in the presence of 11 mM glucose and test substances as indicated and described under Materials and methods. (A,D,F) Changes of TXNIP mRNA levels (ΔΔCt) vs control set to 100% in A and D; vs max effect in F. (B,E,G) Representative western blots of 172 Thr-P-AMPKα and AMPKα1; Tubulin is used as loading control. (C) Quantitative analysis of western blots of three independent experiments of B. Results are given as mean ± SEM of n = 3–4 independent experiments. *p<0.05, **p<0.01, *** p<0.001 denotes significance to control, i.e. 11 mM Glc at 1h; ## p<0.01 to 11 mM Glc at 24 h. $ $p<0.01 significant effect of palmitate in the presence of MS. §§p<0.01 significant effect of palmitate in the presence of MS and BML. Abbreviations: Glc, glucose; Pal, palmitate; MS, MS-275 (HDAC1/2/3 inhibitor); BML, BML-275 (AMPK inhibitor); AICAR, AMPK activator.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Cell Culture, Western Blot, Tandem Mass Spectroscopy

Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: (A-C) INS-1E cells were cultured for 24 h in the presence of 11 mM glucose and test substances as indicated and described under Materials and methods. (A) Percentage of TUNEL positive cells, *p<0.05 significant vs control culture, (B) correlation between TUNEL positive nuclei and relative TXNIP mRNA levels (∆Ct), (C) representative western blot for cleaved caspase 3, GAPDH was used as loading control; (D) representative pictures of INS-1E cells stained with the ROS sensor CellROXGreen. INS-1E cells were cultured for 2 h in the presence of different glucose concentrations, palmitate (600 μM) and menadione (100 μM; ROS inducer) as indicated. CellROXGreen (5 μM) was applied for 1 h in culture. Note the increased green fluorescence (increased ROS levels) in the cells exposed to 30 mM Glc or menadione vs 2.8 mM glucose and 30 mM Glc+palmitate. Abbreviations: Pal, P600, palmitate; BML, BML-275 (AMPK inhibitor); CC-3, cleaved caspase-3.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Cell Culture, TUNEL Assay, Western Blot, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: Transcription of TXNIP is under the control of a protein complex consisting of ChREBP (carbohydrate-responsive element-binding protein) and MondoA as well as the histone acetyltransferase P300. ChREBP is negatively regulated by AMPK. Consequently, stimulation of AMPK by AICAR inhibits ChREBP and TXNIP expression, while inhibition of AMPK by glucose and BML-275 activates ChREBP and increases TXNIP mRNA levels. Histone deacetylase 1 (HDAC1), counteracts P300-mediated histone acetylation, and is involved in insulin-mediated downregulation of TXNIP expression. Thus, inhibition of PI3K, AKT or HDAC1/3 increases TXNIP mRNA levels. Fatty acids-mediated stimulation of insulin secretion occurs via FFAR1/GPR40, a signalling pathway not involved in regulation of TXNIP expression. Fatty acids activate AMPK and have an additional effect on TXNIP mRNA levels. Fatty acids counteract glucose-induced TXNIP expression and ROS elevation, events which do not impede the ER strees-associated lipotoxic effect.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Binding Assay, Expressing, Inhibition, Histone Deacetylase Assay

Journal: Nutrients

Article Title: Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

doi: 10.3390/nu12113326

Figure Lengend Snippet: Activation of AMPK in vitro upregulates bHLH factors, suppresses appetite and increases POMC neuropeptide expression. ( A ) Control NPCs were treated in vitro with AMPK activator (0.5, 1, 2 mM; □) or vehicle (■). NPC protein expression with representative immunoblots of pAMPK, bHLH factors (Mash1, Ngn3) and ( B ) neuropeptides (POMC, AgRP). Values are fold change of vehicle treated ( N = 5; mean ± SE). * p < 0.01 vs control. Abbreviations: AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); POMC (proopiomelanocortin); AgRP (agouti-related protein); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

Article Snippet: To further explore AMPK-mediated effects, NPCs from postnatal 1-day old (p1) Control newborn males were cultured in differentiating medium and treated in vitro with AMPK activator (AICAR; 0.25, 1, 2 mM; #A9978, Sigma-Aldrich, St. Louis, MO, USA), AMPK inhibitor (1, 5, 25 μM Dorsomorphin dihydrochloride, Compound C BML-275,#21207, Cayman Chemicals, Ann Arbor, MI, USA) or vehicle.

Techniques: Activation Assay, In Vitro, Expressing, Control, Western Blot

Journal: Nutrients

Article Title: Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

doi: 10.3390/nu12113326

Figure Lengend Snippet: Suppression of AMPK in vitro downregulates bHLH factors and increases appetite and reduces satiety neuropeptide expression. ( A ) Control NPCs were treated in vitro with AMPK inhibitor (Compound C 1, 5, 25 µm; □) or vehicle (■). NPC protein expression with representative immunoblots of pAMPK, bHLH factors (Mash1, Ngn3) and ( B ) neuropeptides (POMC, AgRP). Values are fold change of vehicle treated ( N = 5; mean ± SE). * p < 0.001 vs control. Abbreviations: Compound C (Dorsomorphin dihydrochloride); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); POMC (proopiomelanocortin); AgRP (agouti-related protein); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

Article Snippet: To further explore AMPK-mediated effects, NPCs from postnatal 1-day old (p1) Control newborn males were cultured in differentiating medium and treated in vitro with AMPK activator (AICAR; 0.25, 1, 2 mM; #A9978, Sigma-Aldrich, St. Louis, MO, USA), AMPK inhibitor (1, 5, 25 μM Dorsomorphin dihydrochloride, Compound C BML-275,#21207, Cayman Chemicals, Ann Arbor, MI, USA) or vehicle.

Techniques: In Vitro, Expressing, Control, Western Blot

Journal: Cancer Science

Article Title: Augmented O ‐Glc NA cylation of AMP ‐activated kinase promotes the proliferation of LoVo cells, a colon cancer cell line

doi: 10.1111/cas.13412

Figure Lengend Snippet: AMP ‐activated kinase ( AMPK ) inhibition accelerated cell growth and inactivated the mTOR pathway in LoVo cells. (a) LoVo cells were cultured in serum‐free DMEM with the indicated concentrations of BML ‐275 for 24 h. Cells were then lysed and subjected to Western blot analysis using anti‐phospho‐ AMPK ( pAMPK ) and phospho‐ mTOR (pm TOR ) antibodies. The total expression levels of AMPK and mTOR were used as controls. Experiments were carried out several times and representative data are shown. Ratios of phosphorylated to total AMPK or mTOR were analyzed statistically with Dunnett's test. * P < 0.05. Molecular weight markers are shown. (b) LoVo cells were cultured with the indicated concentrations of BML ‐275, an AMPK inhibitor, for 24 h. Growth was measured using CCK ‐8. Absorbance rates of the cells with the indicated concentrations of BML ‐275 relative to that of ones without BML ‐275 were analyzed statistically with Steel's test, * P < 0.05.

Article Snippet: Both AICAR and BML‐275 were obtained from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Inhibition, Cell Culture, Western Blot, Expressing, Molecular Weight, CCK-8 Assay