Journal: The Journal of Pathology

Article Title: Hypoxia‐inducible factor 1‐alpha does not regulate osteoclastogenesis but enhances bone resorption activity via prolyl‐4‐hydroxylase 2

doi: 10.1002/path.4906

Figure Lengend Snippet: HIF induction during osteoclast differentiation. (A) Western blot analysis of HIF‐1α protein expression in CD14+ monocytes treated with 25 ng/ml M‐CSF for 2–24 h versus the untreated normoxic control (Nx). Hx = hypoxia (2% O 2 , 24 h). (B) RT‐qPCR comparing HIF1A , HIF2A, LDHA , and SLC2A1 ( GLUT1 ) mRNA on days 3, 5, 7 and 9 of osteoclast differentiation. * p < 0.05; ** p < 0.01. (C) Western blot analysis of HIF‐1α, GLUT1, and LDHA protein on days 3, 5, and 9 of osteoclast differentiation. (D) Normalized HRE‐driven luciferase reporter luminescence on days 3, 5, 7, and 9 of osteoclast differentiation. * p < 0.05; ** p < 0.01.

Article Snippet: Primary antibodies were against HIF‐1α (clone 54, 1:1000; BD Biosciences, Oxford, UK), GLUT1 (ab14683, 1:2500; Abcam, Cambridge, UK), LDHA (NBP1‐48336, 1:2000; Novus Biologicals, Cambridge, UK), and β‐tubulin (clone TUB2.1, 1:2500; Sigma‐Aldrich, Dorset, UK).

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Luciferase

Journal: Acta pharmaceutica Sinica. B

Article Title: The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation.

doi: 10.1016/j.apsb.2023.01.019

Figure Lengend Snippet: Figure 2 Hepatocyte-specific deficiency of NEDD4-1 exacerbates APAP induced hepatotoxicity in vivo and in vitro. (AeD) Serum ALT (A), AST (B), LDH (C) activities, and HMGB1 levels (D) of mice. (E) Representative H&E-stained images (left) and necrotic area quantification (right) of mouse liver sections. Scale bar, 200 mm. (F) Survival rate of Flox (n Z 18) or HepKO (n Z 20) mice treated with 600 mg/kg APAP for 72 h. (G, H) RNA-seq was conducted on total RNA isolated from livers of Flox or HepKO mice treated with 300 mg/kg APAP for 24 h; n Z 3 per group. Volcano plot (G) shows the differentially expressed genes (1201 genes were up-regulated and 736 genes were down-regulated in APAP- treated HepKO mice). GSEA (H) shows the gene sets that the differentially expressed genes significantly enriched. (I) Representative images (left) and quantification (right) of Calcein-AM (green) and PI (red) double-stained primary hepatocytes. Scale bar, 200 mm. (J, K) LDH release (J) and HMGB1 level (K) in the culture medium of primary hepatocytes. Experiments AeE were performed on Flox or HepKO mice treated with saline or 300 mg/kg APAP for 24 h; n Z 5e8 per group. Experiments IeK were performed on primary hepatocytes isolated from Flox or HepKO mice, with vehicle or APAP (10 mmol/L for 12 h) treatment. Data are presented as the mean SEM; *P < 0.05, **P < 0.01.

Article Snippet: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities, and high mobility group box 1 (HMGB1) concentration were measured using the ALT activity assay kit (MAK052; SigmaeAldrich, St. Louis, MO, USA), AST activity assay kit (MAK055; SigmaeAldrich), LDH activity assay kit (MAK066; SigmaeAldrich), and mouse HMGB1 ELISA kit (NBP2-62767; Novus Biologicals, Littleton, CO, USA) according to the manufacturer’s protocols, respectively.

Techniques: In Vivo, In Vitro, Staining, RNA Sequencing, Isolation, Saline

Journal: Acta pharmaceutica Sinica. B

Article Title: The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation.

doi: 10.1016/j.apsb.2023.01.019

Figure Lengend Snippet: Figure 3 Hepatocyte NEDD4-1 overexpression alleviates APAP-induced hepatotoxicity in vivo and in vitro. (A) Representative H&E-stained images (left) and necrotic area quantification (right) of mouse liver sections. Scale bar, 200 mm. (BeE) Serum ALT (B), AST (C), LDH (D) activities, and HMGB1 level (E) of mice. (F) Survival rate of AAV8-Control (n Z 15) or AAV8-NEDD4-1 (n Z 13) infected mice, with 600 mg/kg APAP treated for 72 h. (G) Representative images (left) and quantification (right) of Calcein-AM (green) and PI (red) double-stained primary hepatocytes. Scale bar, 200 mm. (H, I) LDH release (H) and HMGB1 level (I) in the culture medium of primary hepatocytes. Experiments AeE were performed on AAV8-Control or AAV8-NEDD4-1 infected mice, with saline or 300 mg/kg APAP treated for 24 h, n Z 5e8 per group. Experiments GeI were performed on primary hepatocytes transfected with Ad-Vector or Ad-NEDD4-1, with vehicle or APAP (10 mmol/L for 12 h) treatment. Data are presented as the mean SEM; *P < 0.05, **P < 0.01.

Article Snippet: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities, and high mobility group box 1 (HMGB1) concentration were measured using the ALT activity assay kit (MAK052; SigmaeAldrich, St. Louis, MO, USA), AST activity assay kit (MAK055; SigmaeAldrich), LDH activity assay kit (MAK066; SigmaeAldrich), and mouse HMGB1 ELISA kit (NBP2-62767; Novus Biologicals, Littleton, CO, USA) according to the manufacturer’s protocols, respectively.

Techniques: Over Expression, In Vivo, In Vitro, Staining, Control, Infection, Saline, Transfection, Plasmid Preparation

Journal: Acta pharmaceutica Sinica. B

Article Title: The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation.

doi: 10.1016/j.apsb.2023.01.019

Figure Lengend Snippet: Figure 5 Knockdown of VDAC1 mitigates the enhanced AILI events caused by hepatocyte-specific NEDD4-1 deficiency in vivo. (A) Representative Western blots of indicated proteins in the cytosolic and mitochondrial fractions of liver lysates from Flox or HepKO mice treated with 300 mg/kg APAP for 24 h. (B) Representative Western blots of indicated proteins in the cytosolic and mitochondrial fractions of liver lysates from mice treated with saline or 300 mg/kg APAP for 6, 12 or 24 h. (C) Representative IHC staining (left) and quantification (right) in mice treated with saline or 300 mg/kg APAP for 24 h; n Z 3 per group. Scar bar, 200 mm. (D) The hepatic mRNA expressions of Vdac1 in mice treated with saline or 300 mg/kg APAP for 24 h; n Z 8 per group. (E) Representative H&E-stained images (left) and necrotic area quantification (right) of mouse liver sections. Scale bar, 200 mm. (FeI) Serum ALT (F), AST (G), LDH (H) activities, and HMGB1 levels (I) of mice (J) Survival rate of AAV8-shControl (n Z 11) or AAV8-shVDAC1 (n Z 11) infected HepKO mice in response to a lethal dose of APAP (600 mg/kg) for 72 h. (K, L) ATP (K) and ROS (L) contents in the livers of mice. (M) Representative Western blots of indicated proteins in the cytosolic and mitochondrial fractions of liver lysates from AAV8-shControl or AAV8-shVDAC1 infected HepKO mice, with 300 mg/kg APAP treated for 24 h. Experiments EeI, K and L were performed on AAV8-shControl or AAV8-shVDAC1 infected Flox or HepKO mice, with 300 mg/kg APAP treated for 24 h; n Z 5e6 per group. Data are presented as the mean SEM; *P < 0.05, **P < 0.01, n. s., not significant.

Article Snippet: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities, and high mobility group box 1 (HMGB1) concentration were measured using the ALT activity assay kit (MAK052; SigmaeAldrich, St. Louis, MO, USA), AST activity assay kit (MAK055; SigmaeAldrich), LDH activity assay kit (MAK066; SigmaeAldrich), and mouse HMGB1 ELISA kit (NBP2-62767; Novus Biologicals, Littleton, CO, USA) according to the manufacturer’s protocols, respectively.

Techniques: Knockdown, In Vivo, Western Blot, Saline, Immunohistochemistry, Staining, Infection

Journal: Acta pharmaceutica Sinica. B

Article Title: The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation.

doi: 10.1016/j.apsb.2023.01.019

Figure Lengend Snippet: Figure 6 Knockdown of VDAC1 mitigates the enhanced APAP-induced pathological events triggered by hepatocyte-specific NEDD4-1 deficiency in vitro. (A) Representative images (left) and quantification (right) of Calcein-AM (green) and PI (red) double-stained primary he- patocytes. Scale bar, 200 mm. (B, C) LDH release (B) and HMGB1 level (C) in the cell culture medium. (D) Seahorse assays showed the OCR (top) and corresponding statistical results (bottom) in primary hepatocytes. (E) Representative JC-1-stained images (left) and quantification of mitochondria depolarization (right) in primary hepatocytes. Scale bar, 100 mm. (F) Representative MitoSOX Red-stained images (left) and quantification (right) in primary hepatocytes. Scale bar, 200 mm. Experiments AeF were performed on HepKO hepatocytes transfected with Ad- shControl or Ad-shVDAC1, with 10 mmol/L APAP treated for 12 h. Data are presented as the mean SEM; *P < 0.05, **P < 0.01, n. s., not significant.

Article Snippet: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities, and high mobility group box 1 (HMGB1) concentration were measured using the ALT activity assay kit (MAK052; SigmaeAldrich, St. Louis, MO, USA), AST activity assay kit (MAK055; SigmaeAldrich), LDH activity assay kit (MAK066; SigmaeAldrich), and mouse HMGB1 ELISA kit (NBP2-62767; Novus Biologicals, Littleton, CO, USA) according to the manufacturer’s protocols, respectively.

Techniques: Knockdown, In Vitro, Staining, Cell Culture, Transfection

Journal: The Journal of international medical research

Article Title: MYL9 promotes squamous cervical cancer migration and invasion by enhancing aerobic glycolysis.

doi: 10.1177/03000605231208582

Figure Lengend Snippet: Figure 3. MYL9 promoted the progression of SCC by enhancing aerobic glycolysis. (a) MYL9 knockdown decreased the Lactate production of SCC. (b) MYL9 knockdown inhibited the expression of GLUT1, HK2, and LDHA and (c) MYL9 knockdown inactivated the JAK2/STAT3 pathway. MYL9, myosin light chain 9; SCC, squamous cervical cancer; GLUT1, glucose transporter 1; HK2, hexokinase II; LDHA, lactate dehydrogenase A.

Article Snippet: The membranes were blocked with 5% bovine serum albumin in TBS containing Tween-20 (TBST) for 1 hour at 37 C and incubated with primary antibodies againstMYL9 (mouse, 60233-1-Ig, Proteintech, Rosemont, IL, USA), GLUT1 (rabbit, 21829-1-AP, Proteintech), HK2 (mouse, 66974-1-Ig, Proteintech), LDHA (rabbit, 19987-1-AP, Proteintech), b-tubulin (mouse, Proteintech), and GAPDH (mouse, CW0100M, CWBIO, Beijing, China) overnight at 4 C. Following three washes with TBST, the membrane was incubated with horseradish peroxidase-conjugated goat antirabbit or anti-mouse IgG antibody at 37 C for 1 hour.

Techniques: Knockdown, Expressing

Journal: Journal of translational medicine

Article Title: TIMELESS promotes reprogramming of glucose metabolism in oral squamous cell carcinoma.

doi: 10.1186/s12967-023-04791-3

Figure Lengend Snippet: Fig. 5 TIMELESS augments glycolysis via upregulation of key metabolic Molecules. A Correlation analysis between TIMELESS expression and SIRT1, HIF1A and MYC in HNSC tissues form TCGA database. B Correlation analysis between TIMELESS mRNA levels and SIRT1 mRNA levels in 33 OSCC tissues. C qRT-PCR analysis of the expression levels of TIMELESS and key glycolysis genes in OSCC cells. D Western blot analysis of the expression levels of TIMELESS and key glycolysis genes in OSCC cells. E IHC staining of TIMELESS, SIRT1, HK2, PKM2, LDHA and GLUT1 in nude mice tumors. F The transfection efficiency was examined with western blot. G The level of glucose uptake was examined. H Lactate production was examined. I Cell medium pH. J Oxygen consumption level of cell. Data shown were the mean ± S.E.M. from three independent experiments. *P < 0.05; **P < 0.01

Article Snippet: For the immunohistochemistry assays, we used the Ki67 antibody (AF0198) from Affinity Biosciences, and the PCNA antibody (60097-1-Ig), SIRT1 antibody (13161- 1-AP), HK2 antibody (66974-1-Ig), PKM2 antibody (60268-1-Ig), GLUT1 antibody (66290-1-Ig), LDHA antibody (66287-1-Ig), and TIMELESS antibody (14421-1- AP) from Proteintech Group, Inc.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Transfection