Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet:

Article Snippet: For in vitro study, the siRNA targeting TLR2 (siTLR2, 50 nM, KeyGen Biotech, Jiangsu, China) or nontargeting scrambled controls (scRNA, 50 nM, KeyGen Biotech, Jiangsu, China) was applied by Lipocat3000 transfection reagent (Aoqing Biotechnology, Beijing, China).

Techniques: Recombinant, Protein Extraction, Endotoxin Assay, Western Blot, Virus, Software, Real-time Polymerase Chain Reaction, Imaging

Journal: Oncotarget

Article Title: SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism

doi: 10.18632/oncotarget.27486

Figure Lengend Snippet: ( A ) Representation of SLC25A32 genetic alterations across different cancers ( www.cbioportal.org ). ( B ) Spearman´s rank correlation between SLC25A32 mRNA expression (RSEM TPM) and somatic copy number in breast cancer (1075 sample; P < 0.05), ovarian cancer (300 sample; P = 0.0.05) and liver cancer (364 sample; P = 0.05) in patient samples of TCGA. Each dot represents a tumor sample of one particular patient. The dotted line represents a linear regression line and the blue area around the fitted line shows the 95% confidence intervals. ( C ) Median overall survival data of ovarian carcinoma patients with SLC25A32 amplification (67 cases) and no amplification (241 cases). Median survival difference between the two groups is statistically significant ( P = 0.0435). ( D ) Median overall survival data obtained from breast carcinoma patients with SLC25A32 amplification (407 cases) and no amplification (1459 cases) are presented. Median survival difference between the two groups is statistically significant ( P = 0.0000228).

Article Snippet: [2,3,3- 2 H]-Serine tracer was purchased from Buchem BV (Apeldoorn, Netherlands), Lipofectamine RNAiMAX from Life Technologies (Carlsbad, CA, USA) and non-targeting siRNA (scrambled control) and SLC25A32 siRNAs were ordered from Qiagen (Hilden, Germany).

Techniques: Expressing, Amplification

Journal: Oncotarget

Article Title: SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism

doi: 10.18632/oncotarget.27486

Figure Lengend Snippet: ( A , B ) Cells were transfected with either control siRNA oligo (NTC) or SLC25A32 siRNAs (siRNA1 and siRNA2) and real-time cell proliferation of MiaPaCa-2 (A) and PANC-1 (B) cells was measured with the xCELLigence technology over a period of 5 days. Results are presented as mean ± SD of three biological replicates. ( C ) Heat-map representing effect of SLC25A32 knock-down (96 h time-point) on growth inhibition of MiaPaCa-2, MDAM-MB453, TOV21G, DU-145, AGS, OVCAR-8, HCC1143 and PANC-1 cells. Color green and red define sensitive and resistant cells respectively. Proliferation of cells transfected with SLC25A32 siRNA1 and siRNA2 and measured with xCELLigence technology has been normalized to NTC sample and presented as % of cell growth. ( D ) Effect of CRISPR-mediated SLC25A32 knock-out on proliferation of 394 cancer cell line (Achilles Project, Broad Institute). Negative CERES scores indicate more sensitive cell lines.

Article Snippet: [2,3,3- 2 H]-Serine tracer was purchased from Buchem BV (Apeldoorn, Netherlands), Lipofectamine RNAiMAX from Life Technologies (Carlsbad, CA, USA) and non-targeting siRNA (scrambled control) and SLC25A32 siRNAs were ordered from Qiagen (Hilden, Germany).

Techniques: Transfection, Control, Knockdown, Inhibition, CRISPR, Knock-Out

Journal: Oncotarget

Article Title: SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism

doi: 10.18632/oncotarget.27486

Figure Lengend Snippet: ( A ) [2,3,3- 2 H]-serine isotopomer distribution in MiaPaCa-2 cells transfected with either control siRNA oligo (NTC) or SLC25A32 siRNAs (siRNA1 and siRNA2) for 48 h. UN: untreated cells. Results are presented as mean ± SD ( n=3 ), M3 NTC vs. SLC25A32 siRNA2 * P = 0.0318. ( B ) [2,3,3- 2 H]-serine labeling into dTTP in MiaPaCa-2 transfected as in A. Results are presented as mean ± SD ( n = 3 ), M0 NTC vs. SLC25A32 siRNA2 ( ** P = 0.0092); M1 NTC vs. SLC25A32 siRNA2 ( ** P = 0.0113). ( C ) [2,3,3- 2 H]-serine isotopomer distribution in PANC-1 cells transfected as in (A). Results are presented as mean ± SD ( n = 3 ). ( D ) [2,3,3- 2 H]-serine labeling into dTTP in PANC-1 cells transfected with either control siRNA oligo (NTC) or SLC25A32 siRNAs (siRNA1 and siRNA2) for 48 h. Results are presented as mean ± SD ( n = 3 ). ( E ) Cells were transfected with either control siRNA oligo (NTC) or SLC25A32 siRNAs (siRNA1 and siRNA2) and real-time cell proliferation of MiaPaCa-2 cells was measured with the xCELLigence technology. Cells were treated with Formate (1 mM) for rescue experiment. Results are presented as mean ± SD ( n = 3 ). (A–E) Statistical analysis was conducted with Graphpad PRISM (Two-way ANOVA; Bonferroni post-test). For graphs A–D, only comparisons showing statistical significance are represented.

Article Snippet: [2,3,3- 2 H]-Serine tracer was purchased from Buchem BV (Apeldoorn, Netherlands), Lipofectamine RNAiMAX from Life Technologies (Carlsbad, CA, USA) and non-targeting siRNA (scrambled control) and SLC25A32 siRNAs were ordered from Qiagen (Hilden, Germany).

Techniques: Transfection, Control, Labeling

Journal: Oncotarget

Article Title: SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism

doi: 10.18632/oncotarget.27486

Figure Lengend Snippet: ( A , B ) Succinate levels were measure by LC-MS following 72 h SLC25A32 knock-down with siRNA1 and siRNA2 in MiaPaCa-2 (A) and PANC-1 (B) cells. Results for non-targeting control (NTC) and untreated (UN) cells are also presented. Succinate levels were normalized to cell number. (A) NTC vs. SLC25A32 siRNA1, ** P = 0.0013; NTC vs. SLC25A32 siRNA2, **** P ≤ 0.0001. ( C , D ) Oxygen consumption rate (OCR) of MiaPaCa-2 (C) and PANC-1 (D) cells was measured with the Seahorse XF Analyzer following 72 h transfection with non-targeting control (NTC) siRNA, SLC25A32 siRNA1 and SLC25A32 siRNA1. Mitochondrial activity was assessed by consecutive injections of oligomycin (1 μM), FCCP (0.5 μM), and antimycin A (1 μM) or rotenone (1 μM). Results were normalized to cell number and presented as mean ± SD ( n = 3 ), (C) NTC vs. SLC25A32 siRNA1, **** P ≤ 0.0001; NTC vs. SLC25A32 siRNA2, **** P ≤ 0.0001, (D) NTC vs. SLC25A32 siRNA1, ** P = 0.0013; NTC vs. SLC25A32 siRNA2, **** P ≤ 0.0001. ( E ) OCR measurement in MiaPaCa-2 cells was conducted as in C. Cells were treated with Riboflavin (10 μM) for rescue experiment. SLC25A32 siRNA1 untreated vs. riboflavin, *** P = 0.0013; SLC25A32 siRNA2 untreated vs. riboflavin, **** P ≤ 0.0001. ( F ) Bar graph of maximal respiration capacity from experiment conducted in E. Data presented is extracted from second time-point following FCCP injection. Results were normalized to cell number and presented as mean ± SD ( n = 3 ). ( G ) Cells were transfected with either control siRNA oligo (NTC) or SLC25A32 siRNAs (siRNA1 and siRNA2) and real-time cell proliferation of MiaPaCa-2 cells was measured with the xCELLigence technology. Cells were treated with Riboflavin (10 μM) for rescue experiment. Results are presented as mean ± SD ( n = 3 ). SLC25A32 siRNA1 untreated vs. riboflavin, * P = 0.0217; SLC25A32 siRNA2 untreated vs. riboflavin, ** P = 0.0031. (A–G) Two-way ANOVA and Bonferroni post-test were used for statistical analysis with Graphpad-Prism6.

Article Snippet: [2,3,3- 2 H]-Serine tracer was purchased from Buchem BV (Apeldoorn, Netherlands), Lipofectamine RNAiMAX from Life Technologies (Carlsbad, CA, USA) and non-targeting siRNA (scrambled control) and SLC25A32 siRNAs were ordered from Qiagen (Hilden, Germany).

Techniques: Liquid Chromatography with Mass Spectroscopy, Knockdown, Control, Transfection, Activity Assay, Injection

Journal: Oncotarget

Article Title: SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism

doi: 10.18632/oncotarget.27486

Figure Lengend Snippet: ( A ) MiaPaCa-2 and PANC-1 cells were transfected with siRNAs targeting SLC25A32 (siRNA1 and 2) and a non-targeting control (NTC) for 72 h. Cells were stained with Mitosox red dye (5 μM) for 30 min and superoxide levels were detected by flow cytometry. Results are presented as mean ± SD, relative to NTC. MiaPaCa-2 cells, NTC vs. SLC25A32 siRNA 1, * P = 0.0494 and NTC vs. SLC25A32 siRNA 2, * P = 0.0224. ( B ) MiaPaCa-2 cells were transfected as in (A). Proteins were extracted and resolved by Immunoblotting. Membranes were incubated with the indicated antibodies. Calnexin was used as loading control. ( C ) MiaPaCa-2 and PANC-1 cells were transfected as in (A). Cells were stained with H 2 DCFDA dye (5 μM) for 30 min and hydrogen peroxide levels were detected by flow cytometry. Results are presented as mean ± SD, relative to NTC. ( D ) MiaPaCa-2 were transfected as in (A). Cells were treated with cell permeable glutathione (GSH 100 μM) and stained with H 2 DCFDA dye (5 μM) for 30 min and hydrogen peroxide levels were detected by flow cytometry. Results are presented as mean ± SD, relative to NTC. SCLC25A32 siRNA 1 untreated vs. GSH, *** P = 0.0001 and SCLC25A32 siRNA 2 untreated vs. GSH, *** P = 0.0001. ( E ) MiaPaCa-2 cells were transfected as in A. Ratio of reduced (GSH) to oxidized glutathione (GSSG) was measured by LC-MS. Results are presented as mean ± SD ( n=3 ). MiaPaCa-2 NTC vs. SLC25A32 siRNA2, * P = 0.0201. (A–E) Two-way ANOVA and Bonferroni post-test were used for statistical analysis with Graphpad-Prism6.

Article Snippet: [2,3,3- 2 H]-Serine tracer was purchased from Buchem BV (Apeldoorn, Netherlands), Lipofectamine RNAiMAX from Life Technologies (Carlsbad, CA, USA) and non-targeting siRNA (scrambled control) and SLC25A32 siRNAs were ordered from Qiagen (Hilden, Germany).

Techniques: Transfection, Control, Staining, Flow Cytometry, Western Blot, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism

doi: 10.18632/oncotarget.27486

Figure Lengend Snippet: ( A , B ) MiaPaCa-2 (A) and PANC-1 (B) cells were transfected with siRNAs targeting SLC25A32 (siRNA1 and 2) and a non-targeting control (NTC) for 72 h and treated with L-Buthionine sulfoximine (BSO 10 μM). Cells were stained with H 2 DCFDA dye (5 μM) for 30 min and hydrogen peroxide levels were detected by flow cytometry. Results are presented as mean ± SD, relative to NTC. (A) NTC untreated vs. BSO, ** P = 0.0030, SLC25A32 siRNA1 untreated vs. BSO, *** P = 0.0002, SLC25A32 siRNA2 untreated vs. BSO, *** P = 0.0001 ; (B) NTC untreated vs. BSO, * P = 0.0385, SLC25A32 siRNA2 untreated vs. BSO, *** P = 0.0050. ( C ) Cell growth of MiaPaCa-2 and PANC-1 cells transfected with siRNAs targeting SLC25A32 (siRNA1 and 2) and a non-targeting control (NTC) was measured with the XCELLigence technology. At the time of transfection, BSO 10 μM was added to cells. Data from the 96 h time-point were normalized to NTC control and presented as mean ± SD ( n = 3 ). MiaPaCa-2, SLC25A32 siRNA2 untreated vs. BSO, *** P = 0.0002. ( D ) Cell growth of MiaPaCa-2 cells transfected with siRNAs targeting SLC25A32 (siRNA1 and 2) and a non-targeting control (NTC) was measured with the XCELLigence technology. At the time of transfection, reduced glutathione (GSH 100 μM) was also added to cells. Results are normalized to NTC control and presented as mean ± SD ( n = 3 ). SLC25A32 siRNA1 untreated vs. GSH, **** P ≤ 0.0001, SLC25A32 siRNA2 untreated vs. GSH, **** P ≤ 0.0001. (A–D) Two-way ANOVA and Bonferroni post-test were used for statistical analysis with Graphpad-Prism6.

Article Snippet: [2,3,3- 2 H]-Serine tracer was purchased from Buchem BV (Apeldoorn, Netherlands), Lipofectamine RNAiMAX from Life Technologies (Carlsbad, CA, USA) and non-targeting siRNA (scrambled control) and SLC25A32 siRNAs were ordered from Qiagen (Hilden, Germany).

Techniques: Transfection, Control, Staining, Flow Cytometry