Journal: International Journal of Molecular Sciences

Article Title: Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1β mRNA Expression

doi: 10.3390/ijms23105331

Figure Lengend Snippet: Compounds 5f and 4d attenuate inflammation in vitro. Effects of compounds (10 µM) on the protein levels of STAT3, IκB, and NF-κB in AML-12 cells. ( a ) Expression levels of inflammation related-proteins in vitro. ( b – d ) Protein expression levels were normal-ized against the indicated protein. * p < 0.05 compared with the group treated with the PBS (vehicle). + p < 0.05 compared with the group treated with the LPS. Data are pre-sented as the mean ± SD.

Article Snippet: The human keratinocytes HaCaT or the alpha mouse liver 12 (AML-12) cells were obtained from Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: In Vitro, Expressing

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: AMPK attenuates alcohol-induced liver injury and mitochondrial injury. ( A-C, F ) Analysis of mice on pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). ( D, E, G ) Analysis of AML-12 cells on control, 200 mM ethanol-treated for 24 h (Eth), 200 mM ethanol + 3.2 mM metformin-treated for 24 h (Eth+ Met). (A) Electron microscopy analyses of mice liver display mitochondria with normal morphology, destroyed cristae structure, and fewer mitochondrial number. (B) Quantification of mitochondrial number in 200 μm 2 (scale bar: 1 μm). (C) qRT-PCR was used to identify mtDNA level in mice serum, which can indicate mitochondrial injury level in mice hepatocytes. (D) AML-12 cells were stained with MTRC to identify the relative mtROS level. The fluorescence light intensity was measured by a multi-function micro reader. (E) AML-12 cells were stained with MTRC, and images were captured with a fluorescence microscope (scale bar: 50 μm) Arrow indicates mtROS points. (F, G) Western blot results show LC3-II:LC3-I ratio and SQSTM1 level in mice liver and AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Control, Electron Microscopy, Quantitative RT-PCR, Staining, Fluorescence, Microscopy, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: AMPK upregulates mitophagy level in mice and AML-12 cells. ( A-C, G ) Analysis of AML-12 cells on control, 200 mM ethanol-treated for 24 h (Eth), 200 mM ethanol + 3.2 mM metformin-treated for 24 h (Eth+ Met). (D-F) Analysis of mice on pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). (A) AML-12 cells were transfected with mito timer plasmid in advance (6 h), and were treated with ethanol, metformin (24 h). A multi-function micro reader was used to detect the 500:583 nm (green:red) fluorescence signal ratio. (B) AML-12 cells were treated with ethanol, metformin for 24 h, and were stained with MTDR for 15 min. A multi-function micro reader was used to detect 665 nm fluorescence light intensity. (C) FCM (flow cytometry) experiments and analysis. AML-12 cells were transfected with mtKeima plasmid in advance (6 h), and were treated with ethanol, metformin (24 h). Analysis of mitophagic ratio in Con, Eth, and Eth+Met cells. (D) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. (E) Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (F, G) Western blot results show the protein (PINK1, PRKN) level in mitochondrial lysates of mice livers and AML-12 cells. TOMM20 was used as an internal reference in mitochondrial lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Control, Transfection, Plasmid Preparation, Fluorescence, Staining, Flow Cytometry, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: AMPK upregulates UQCRC2 protein level (Complex III activity) in ALD. (A, C) Analysis of mice on pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). (B, D) Analysis of AML-12 cells on control, 200 mM ethanol (Eth), 200 mM ethanol + 3.2 mM metformin (Eth+ Met). (A, B) Western blot results: Ethanol caused the low expression of UQCRC2 protein in liver and AML-12 cells, whereas metformin can significantly enhance UQCRC2 protein level. (C, D) Mitochondrial activity assay: Ethanol reduced complex III activity, whereas metformin can upregulate complex III activity in 10 days ethanol-fed mice or ethanol-treated AML-12 cells. (E) IHC results of UQCRC2 in human samples, and IHC scores analysis. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Activity Assay, Control, Western Blot, Expressing

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: AMPK indirectly regulates UQCRC2. (A) qRT-PCR results of Uqcrc2 mRNA fold in pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). (B) A heatmap showed differentially expressed RNA with the same expression pattern in pair-fed and 10-d eth-fed mice. (C) Nfe2l2 fold change (RNA-seq results). (D) Gene promoter analysis: Three sites in Uqcrc2 promoter that NFE2L2 can bind. (E) Luciferase reporter gene results in AML-12 cells. AML-12 cells were transfected with NC 1 (pGL3-basic) + NC 2 (pcDNA3.1), NC 1 + pGL3- Nfe2l2 , NC 2 + pcDNA3.1- Uqcrc2 , pGL3- Nfe2l2 + pcDNA3.1- Uqcrc2 . Data are presented as the ratio of Renilla luciferase activity to firefly activity (n = 3). (F) qRT-PCR results of Nfe2l2 mRNA fold in pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). (G) ChIP assay showing the interaction of NFE2L2 with the potential binding site on the Uqcrc2 promoter in AML-12 cells. Metformin enhanced UQCRC2 fold number in the anti- NFE2L2 group, whereas shPrkaa1 reduced metformin-induced high enrichment of UQCRC2 in the anti-NFE2L2 group. (H, I) Analysis of AML-12 cells on NC (negative control), NC+ 3.2 Metformin (Met), 3.2 mM Met + shPrkaa1 (50 nM), NC+ ethanol, NC+ethanol + metformin, Ethanol + Metformin + shPrkaa1 . Western blot results show NFE2L2 and UQCRC2 protein levels in AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Quantitative RT-PCR, Expressing, RNA Sequencing, Luciferase, Transfection, Activity Assay, Binding Assay, Negative Control, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: The effects of UQCRC2 genetic intervention in mice and AML-12 cells. ( A- E, I- K ) Analysis of mice on pair-fed mice was injected with AAV-NC or lentivirus-NC (n = 15), pair-fed mice were injected with AAV-UQCRC2 OE (n = 15), or pair-fed mice were injected with AAV-UQCRC2 KD (n = 15). (F-H) Analysis of AML-12 cells on NC plasmid (50 nM), UQCRC2 OE plasmid (50 nM), UQCRC2 KD plasmid (50 nM). (A, B) Test of GPT, and GOT1 levels in serum. (C) HE staining results and analysis: UQCRC2 OE or KD cannot cause liver injury (scale bar: 200 μm). (D) Oil red staining results and analysis: UQCRC2 OE or KD cannot influence lipid accumulation in mice liver (scale bar: 100 μm). (E). qRT-PCR was used to identify the mtDNA level in mice serum. (F) AML-12 cells were stained with MTRC to identify the relative mtROS level. The fluorescence light intensity was measured by a multi-function microplate reader. (G) AML-12 cells were treated with ethanol, metformin for 24 h, and were stained with MTDR for 15 min. A multi-function micro reader was used to detect 665 nm fluorescence light intensity. (H) FCM (flow cytometry) experiments and analysis. AML-12 cells were transfected with mtKeima plasmid in advance (6 h), and were treated with ethanol, metformin (24 h). Analysis of mitophagic ratio in NC, UQCRC2 OE, and UQCRC2 KD cells. (I) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (J, K) Western blot results show the protein (PINK1, PRKN) level in mice livers. TOMM20 was used as an internal reference in mitochondrial lysates. WB results show LC3-II:LC3-I ratio in mice liver. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Injection, Plasmid Preparation, Staining, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Transfection, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: UQCRC2 intervention regulates mitophagy in AML-12 cells. (A) OCR in NC and UQCRC2 KD AML-12 cells. (B) ATP production level in NC and UQCRC2 KD AML-12 cells. (C) JC-1 staining results of NC and UQCRC2 KD AML-12 cells. The ratio of absorbance 585:529 is ΔΨ. (D) OCR in NC, 10 μM CCCP, and 10 μM CCCP+UQCRC2 KD AML-12 cells. (E) ATP production level in NC, 10 μM CCCP, and 10 μM CCCP+UQCRC2 KD AML-12 cells. (F) Western blot results show the LC3 II/LC3 I ratio in AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. (G) Western blot results show the p-AMPK level in NC and UQCRC2 KD AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. (H) Western blot results show the PINK1 level in NC and PINK1 KD AML-12 cells, (I) and PRKN protein level on mitochondria in NC+CCCP, PINK1 KD+CCCP, and PINK1 KD+ UQCRC2 OE+CCCP AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. TOMM20 was used as an internal reference in mitochondrial lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Staining, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: UQCRC2 OE or KD can attenuate or deteriorate ALD through mitophagy. ( A-E, H, I, K ) Analysis of mice on 10-d Eth-fed mice was injected with AAV-NC or lentivirus-NC (n = 15), 10-d Eth-fed mice were injected with AAV-UQCRC2 OE (n = 15), or 10-d Eth-fed were injected with AAV-UQCRC2 KD (n = 15). ( F, G, J ) Analysis of AML-12 cells on 200 mM Eth + NC plasmid (50 nM), Eth+ UQCRC2 OE plasmid (50 nM), Eth+ UQCRC2 KD plasmid (50 nM). (A, B) Test of GPT, and GOT1 level in serum. (C) HE staining results and analysis (scale bar: 200 μm). (D) Oil red staining results and analysis (scale bar: 100 μm). (E) qRT-PCR was used to identify the mtDNA level in mice serum. (F) AML-12 cells were stained with MTRC to identify the relative mtROS level. The fluorescence light intensity was measured by a multi-function micro reader. (G) AML-12 cells were treated were stained with MTDR for 15 min. A multi-function micro reader was used to detect 665 nm fluorescence light intensity. (H, I) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (J) FCM (flow cytometry) experiments and analysis. AML-12 cells were transfected with mtKeima plasmid in advance (6 h), and were detected by FCM. Analysis of mitophagic ratio in NC+Eth, Eth+UQCRC2 OE, and Eth+UQCRC2 KD cells. (K) Western blot results showing the protein (PINK1, PRKN) level in mice livers. TOMM20 was used as an internal reference in mitochondrial lysates. Western blot results show LC3-II:LC3-I ratio in mice liver. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Injection, Plasmid Preparation, Staining, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Transfection, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: UQCRC2 protein is required for AMPK regulating mitophagy in ALD. ( A-E, I-K ) Analysis of mice on 10 d Eth-fed mice was injected with AAV-NC (n = 15), 10-d Eth-fed mice were orally administrated with 150 mg/kg metformin (n = 15), or 10-d Eth-fed were orally administrated with 150 mg/kg metformin and injected with AAV-UQCRC2 KD (n = 15). (F-H) Analysis of AML-12 cells on 200 mM Eth + NC plasmid (50 nM), Eth+ NC plasmid + 3.2 mM metformin, Eth+ UQCRC2 KD plasmid (50 nM) +metformin. (A, B) Test of GPT and GOT1 level in serum. (C) HE staining results and analysis (scale bar: 200 μm). (D) Oil red staining results and analysis (scale bar: 100 μm). (E) qRT-PCR was used to identify the mtDNA level in mice serum. (F) AML-12 cells were stained with MTRC to identify the relative mtROS level. The fluorescence light intensity was measured by a multi-function micro reader. (G) AML-12 cells were treated were stained with MTDR for 15 min, and were measured by a multi-function microplate reader. (H) FCM (flow cytometry) experiments and analysis. Analysis of mitophagic ratio in AML-12 cells. (I) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (J, K) Western blot results showing the protein (PINK1, PRKN) level in mice livers. TOMM20 was used as an internal reference in mitochondrial lysates. WB results show LC3-II:LC3-I ratio in mice liver. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Injection, Plasmid Preparation, Staining, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: PRKN protein is downstream of UQCRC2 in the mitophagy signaling pathway. ( A-E, H-J ) Analysis of mice on 10-d Eth-fed mice was injected with AAV-NC(n = 15), 10-d Eth-fed mice were injected with AAV-UQCRC2 KD (n = 15), or 10-d Eth-fed mice were injected with AAV-UQCRC2 KD and AAV- PRKN OE (n = 15). ( F, G, J ) Analysis of AML-12 cells on 200 mM Eth, Eth+ UQCRC2 KD plasmid (50 nM) + PRKN OE plasmid (50 nM). (A, B). Test of GPT, and GOT1 level in serum. (C). HE staining results and analysis (scale bar: 200 μm). (D). Oil red staining results and analysis (scale bar: 100 μm). (E). qRT-PCR was used to identify the mtDNA level in mice serum. (F). AML-12 cells were stained with MTRC to identify the relative mtROS level. (G). AML-12 cells were treated were stained with MTDR for 15 min, and were measured by micro fluorescence reader at 665 nm. (H, I) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (J). WB results show LC3-II:LC3-I ratio in mice liver and AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Injection, Plasmid Preparation, Staining, Quantitative RT-PCR, Fluorescence