Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: CD38 is closely related to HNSC immune microenvironment.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques:

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: CD38 and the number of immune cells in the HNSC microenvironment correlate positively. The TIMER database was used to analyze the correlation between the expression of CD38 in HNSC and the expression of biomarkers in immune infiltrating cells. (A-K) Scatter plots representing correlation between CD38 expression and the biomarkers of (A) B cells (CD19 and CD79A); (B) Monocytes (CD86); (C) CD8+ T cells (CD8A and CD8B); (D) Neutrophils (CCR7 and ITGAM); (E) Tregs (CCR8 and STAT5B); (F) TAMs (CD68 and IL10); (G) Exhausted T cells (CTLA4, HAVCR2 and PDCD1); (H) M1 Macrophages (IRF5); (I) M2 Macrophages (CD163, MS4A4A and VSIG4); and (J) Th1 cells (IFNG, STAT1, and TBX21)) in HNSC samples. (K) Th2 cells (STAT6).

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: CD38 mRNA level of in head and neck squamous cell carcinoma . Overexpression gene rank, fold change, and related p -values are shown, based on Oncomine 4.5 analysis. (A-D) Box plot showing CD38 mRNA expression in Ginos Head-Neck, Pyeon Multi-cancer, Toruner Head-Neck, and Cromer Head-Neck datasets, respectively.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Over Expression, Expressing

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Relationship between CD38 expression and clinical parameters in healthy subjects and patients with HNSC. (A) Box diagram showing the relative transcript levels of CD38 in HNSC and healthy samples. (B) Relative CD38 transcription level in healthy individuals or in patient with HNSC in disease stage 1, 2, 3, or 4 shown as a box plot. (C) Relative CD38 transcription level in healthy individuals of any race or in patients with HNSC of Caucasian, African-American, or Asian ethnicity, shown using a box plot. (D) Relative C D38 transcription level in healthy individuals of either gender or in female or male patients with HNSC, shown as a box plot. (E) Relative CD38 transcription level in healthy individuals with any age or in patients with HNSC at ages of 21-40, 41-60, 61-80, or 81-100 years, shown as a box plot. (F) Relative CD38 transcription level in healthy individuals or in patients with HNSC with tumor grade 1, 2, 3, or 4, as shown in a box plot. Data are the mean ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Relationship between prognosis and CD38 expression in patients with HNSC. (A) The relationship between the expression of CD38 and OS (overall survival) in patients with HNSC. (B) Relationship between the tumor grade and CD38 in different clinical tumors of HNSC and the OS in patients with HNSC.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Analysis of the correlation between CD38 in HNSC and biomarkers of immune cells (TIMER)

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques:

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Activation of the PI3K pathway induced by overexpression of CD38. Western blotting showed that the level of PD-L1 (A) and PI3K phosphorylated at Tyr607 and Ser605 (C,D) and were increased, while the level of PTEN (B) was decreased after overexpression of CD38.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Activation Assay, Over Expression, Western Blot

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Expression of CD38 in nasopharyngeal carcinoma cell lines. (A) Western blotting analysis showing the levels of CD38 in nasopharyngeal carcinoma epithelial cells (NP69) and different nasopharyngeal carcinoma cell lines (CNE2, HNE2, 6-10B, HONE1, HK1, and C666-1). (B) Detection of CD38 levels in radiotherapy-tolerant cell lines HNE2-IR and CNE2-IR. (C) HNE2 and CNE2 cells were both transfected with the CD38 expression vector and control vector, CD38 levels were detected using immunoblotting. (D,E) Clone formation experiment to verify the successful construction of HNE2-IR and CNE2-IR. The experiments were performed in triplicate and repeated at least two times. Student's t test was used for statistical analysis. * P < 0.01; ** P < 0.001.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of Cancer

Article Title: Integrative Analysis Identified CD38 As a Key Node That Correlates Highly with Immunophenotype, Chemoradiotherapy Resistance, And Prognosis of Head and Neck Cancer

doi: 10.7150/jca.59730

Figure Lengend Snippet: Resistance in CD38-high cells to radiation treatment at 4 Gy and 6 Gy. (A,B) The effects of CD38 overexpression on colony formation in HNE2 and CNE2 cells under radiation dose of 0, 2, 4, and 6 Gy. The experiments were performed in triplicate and repeated at least three times. Student's t test was used for statistical analysis. **, P < 0.01; ***, P < 0.001.

Article Snippet: Then, 50 μg lysate was electrophoresed on 10% separation gel and transferred to a polyvinylidene fluoride (PVDF) membrane (HyClone Laboratories, Logan, UT, USA), which was blocked with 5% non-fat milk diluted in phosphate-buffered saline (PBS)-Tween20 for approximately 2 h. Primary antibody solution was then added and incubated at 4 °C for 12 h. The primary antibodies used were: Anti-phospho-phosphatidylinositol 3-kinase (PI3K) p85 alpha (Tyr607), anti-phospho-PI3 Kinase p85 beta (Ser605), anti‑phospho‑PI3K p85 (Tyr458), anti-CD38, anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN), anti-NF-κb, anti-mTOR, anti-p53, and anti-PD-L1 (at dilution ratios of 1:500-1:2000 using 5% Skim milk, Affinity Biosciences (Cincinnati OH, USA) supplied the antibodies for PI3K, CD38, PTEN, and Cell Signaling Technology (Danvers, MA, USA) supplied the PD-L1 antibodies.

Techniques: Over Expression

Journal: Cells

Article Title: Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice

doi: 10.3390/cells11233812

Figure Lengend Snippet: qPCR primers.

Article Snippet: The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

Techniques: Sequencing

Journal: Cells

Article Title: Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice

doi: 10.3390/cells11233812

Figure Lengend Snippet: Cold exposure increases NAD(H) levels and decreases NADPH levels in liver from WT mice. Livers were harvested from WT and Cd38 −/− mice housed at 30 °C for 7 days (30 °C, black bars), or at 22 °C for 7 days (22 °C, grey bars), or at 22 °C for 6 days and at 6 °C for 1 day (6 °C, white bars). For dinucleotide content assay, tissues were minced on ice and then deproteinized in PCA or NaOH. Acid-extracted samples were used to evaluate NAD + and NADP + ( A , C ), whereas base-extracted samples were used to determine NADH and NADPH levels ( B , D ). Results are the means ± SD ( n = 6–7). Data analyzed by ANOVA with Tukey’s test: *, p < 0.05; **, p < 0.01; data analyzed by Student’s t -test: # , p < 0.05; ## , p < 0.01; ### , p < 0.001; & , p < 0.00001 compared with the corresponding WT.

Article Snippet: The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

Techniques:

Journal: Cells

Article Title: Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice

doi: 10.3390/cells11233812

Figure Lengend Snippet: Cold exposure down-regulates CD38 levels in liver from WT mice. Livers were harvested from WT and Cd38 −/− mice housed at 30 °C for 7 days (30 °C, black bars), or at 22 °C for 7 days (22 °C, grey bars), or at 22 °C for 6 days and at 6 °C for 1 day (6 °C, white bars). ( A – C ), CD38 expression was measured by RT-PCR ( A ), Western blot analyses ( B ), and by evaluating the enzymatic activity and measuring ADPR production starting from NAD + ( C ). ( D , G ) Nampt and Fgf21 gene expression levels were evaluated by RT-PCR. ( E , F ), NAD + synthesis was evaluated measuring NAD + production starting from NAM, PRPP, and ATP ( E ), or starting from NMN and ATP ( F ). Results are the means ± SD ( n = 4–5). Data analyzed by ANOVA with Tukey’s test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; data analyzed by Student’s t -test: ### , p < 0.001; #### , p < 0.0001; & , p < 0.00001, compared with the corresponding WT.

Article Snippet: The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay

Journal: Cells

Article Title: Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice

doi: 10.3390/cells11233812

Figure Lengend Snippet: G6PD and malic enzyme are downregulated in liver of WT and Cd38 −/− mice. Livers were harvested from WT and Cd38 −/− mice housed at 30 °C for 7 days (30 °C, black bars), or at 22 °C for 7 days (22 °C, grey bars), or at 22 °C for 6 days and at 6 °C for 1 day (6 °C, white bars). Nadk , G6pdx and Me1 expressions were measured by RT-PCR ( A , C , E ). NADK activity was evaluated by measuring the NADP + production, starting from NAD + and ATP ( B ). G6PD activity was evaluated by measuring NADPH production, starting from G6P and NADP + ( D ). Results are the means ± SD ( n = 4–5). Data analyzed by ANOVA with Tukey’s test: *, p < 0.05; **, p < 0.01; ****, p < 0.0001; data analyzed by Student’s t -test: ### , p < 0.001; #### , p < 0.0001 compared with the corresponding WT.

Article Snippet: The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

Techniques: Reverse Transcription Polymerase Chain Reaction, Activity Assay

Journal: Cells

Article Title: Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice

doi: 10.3390/cells11233812

Figure Lengend Snippet: Cold exposure determines a metabolic switch from glycolysis to gluconeogenesis in WT mice but not in mice lacking Cd38 −/− . Livers collected from WT and Cd38 −/− mice housed at 30 °C for 7 days (30 °C, black bars), or at 22 °C for 7 days (22 °C, grey bars), or at 22 °C for 6 days and at 6 °C for 1 day (6 °C, white bars) were analyzed for gene expression and enzymatic activity quantification. Pfk1 , Gapdh , and G6pase gene expression levels were measured by RT-PCR ( A , B , F ). GAPDH protein expression levels were measured by Western blot analyses ( C , D ). LDH activity was measured by quantifying NADH-produced incubating liver lysates with NAD + and L(+)-lactate ( E ). G6Pase activity was evaluated as G6P depletion from the incubation with liver lysates ( G ). G6P levels were measured in acid-extracted samples, using a cycling assay ( H ). Hepatic glycogen was measured in fresh minced tissues by glucose quantification, upon glycogen hydrolysis, using a commercially available kit ( I ). All results are the means ± SD of 4–5 independent determinations for each condition. Data analyzed by ANOVA with Tukey’s test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; data analyzed by Student’s t -test: # , p < 0.05; ## , p < 0.01; ### , p < 0.001 compared with the corresponding WT.

Article Snippet: The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Produced, Incubation

Journal: Cells

Article Title: Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice

doi: 10.3390/cells11233812

Figure Lengend Snippet: Cold exposure SIRT3 activity in WT, but not in Cd38 −/− mice, with SIRT3 over-activated at warm temperatures in Cd38 −/− mice. Livers from WT and Cd38 −/− mice, kept for 1 week at 30 °C (black bars) or 6 days at 22 °C followed by 1 day at 6 °C (white bars), were used. Pdh expression was evaluated by RT-PCR ( A ). SIRT3 activity was evaluated by Western blot, measuring the acetylation level of SOD2, a substrate of SIRT3 activity ( B – E ). Results of quantification are the mean ± SD from n = 3 animals for each condition. Data analyzed by ANOVA with Tukey’s test: *, p < 0.05; **, p < 0.01; data analyzed by Student’s t -test: # , p < 0.05; & , p < 0.00001 compared with the corresponding WT.

Article Snippet: The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

Techniques: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cells

Article Title: Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice

doi: 10.3390/cells11233812

Figure Lengend Snippet: Changes in lipid metabolism induced by cold in WT and Cd38 −/− mice. Livers collected from WT and Cd38 −/− mice housed at 30 °C for 7 days (30 °C, black bars), or at 22 °C for 7 days (22 °C, grey bars), or at 22 °C for 6 days and at 6 °C for 1 day (6 °C, white bars) were analyzed for gene expression. Acaca and Fasn expression levels were measured by RT-PCR ( A , B ). Total TGs and free glycerol were detected in freshly chopped livers by quantitative fluorometric assay, using a commercially available kit ( C , D ). Blood samples from each mouse were spun down and the resulting serum were used to quantify TGs, cholesterol, and BAs ( E , F ). Results are the mean ± SD ( n = 4–5). Data analyzed by ANOVA with Tukey’s test: *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001; data analyzed by Student’s t -test: # , p < 0.05, ## , p < 0.01, ### , p < 0.001, compared with the corresponding WT.

Article Snippet: The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice

doi: 10.3390/cells11233812

Figure Lengend Snippet: Scheme of cold-induced changes in liver from WT and Cd38 −/− mice. During cold exposure, CD38 expression is downregulated, and NAD(H) levels are increased. The pentose phosphate pathway (PPP) is downregulated, and NADPH levels are decreased, both in WT and Cd38 −/− mice, in agreement with a reduced fatty acid (FA) synthesis. The glycolytic pathway is also downregulated in WT mice, re-directing glucose-6-P (G6P) towards glucose release. Conversely, glycolysis is not downregulated in Cd38 −/− mice, indicating that CD38 is important for the glycolysis/gluconeogenesis cross-regulation. Sirtuin 3 activity, controlling mitochondrial metabolism, is enhanced by cold exposure in liver of WT mice, whereas it is upregulated in Cd38 −/− mice, regardless of the temperature.

Article Snippet: The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

Techniques: Expressing, Activity Assay