Journal: Prostaglandins, leukotrienes, and essential fatty acids

Article Title: Resolvin D4 mitigates lipopolysaccharide-induced lung injury in mice.

doi: 10.1016/j.plefa.2024.102652

Figure Lengend Snippet: Fig. 3. The effect of RvD4 administration on the cell apoptosis of alveolar epithelial cells and the protein expression of Zo-1 and Sirt3 in the lungs of LPS-injected mice. A: Representative profiles of TUNEL staining (red) of lung sections 24 h after LPS injection (5 mg/kg). Note that 4′,6-diamidino-2-phenylindole (DAPI) was used in the counterstaining of the nucleus (blue). Scale bars: 50 μm. Three high-power fields per slide were randomly selected, and the number of TUNEL-positive cells and DAPI-positive cells were counted. The percentages of TUNEL-positive cells were obtained by dividing the number of TUNEL-positive cells by the number of DAPI- positive cells. TUNEL-positive cells (arrows) were visualized in lung tissues of LPS-injected mice. Data are means ± SEM of 4 mice per group. *p < 0.05. B,C: Immunoblots of Zonula occludens-1 (Zo-1) and sirtuin-3 (Sirt3) from the PBS control, LPS/saline, and LPS/RvD4 groups. The quantitative comparisons were per formed using β-actin as the loading control (PBS control, n = 7; LPS/saline, n = 6; LPS/RvD4, n = 8). Values are mean ± SEM, analyzed by one-way ANOVA and then Dunnett multiple comparison test. *p < 0.05 vs. the LPS/saline group.

Article Snippet: We measured the expression levels of proteins in the lysates of lung tissue by western blotting, using antibodies recognizing the following proteins: β-actin (Sigma-Aldrich Japan), Zonula occludens-1 (Zo-1) (Cell Signaling Technology Japan, Tokyo), and sirtuin-3 (Sirt3) (Cell Signaling Technology Japan).

Techniques: Expressing, Injection, TUNEL Assay, Staining, Western Blot, Control, Saline, Comparison

Journal: Prostaglandins, leukotrienes, and essential fatty acids

Article Title: Resolvin D4 mitigates lipopolysaccharide-induced lung injury in mice.

doi: 10.1016/j.plefa.2024.102652

Figure Lengend Snippet: Fig. 5. The silencing of Sirtuin-3 (SIRT3) abolished the anti-inflammatory effect and retention of cell integrity in RvD4-treated BEAS-2B cells. A: The silencing efficiency of SIRT3 was determined by quantitative real-time PCR. Data are shown as mean ± SEM (n= 4 per group). *p < 0.05. B: The mRNA expression levels of IL- 1β and IL-6 in BEAS-2B cells transfected with SIRT3 siRNA (si-SIRT3) or scrambled siRNA (si-scr) at 3 h after LPS stimulation are shown. Gene expressions were normalized with hypoxanthine phosphoribosyltransferase 1 (HPRT1). Data are shown as mean ± SEM (n= 3 per group). *p < 0.05. n.s.: not significant. C: Time- dependent changes in the TER from baseline in BEAS-2B cells exposed to PBS or LPS. Data are shown as mean ± SEM, analyzed by one-way ANOVA and then Dunnett multiple comparison test (n= 4 per group). *p < 0.05. n.s.: not significant.

Article Snippet: We measured the expression levels of proteins in the lysates of lung tissue by western blotting, using antibodies recognizing the following proteins: β-actin (Sigma-Aldrich Japan), Zonula occludens-1 (Zo-1) (Cell Signaling Technology Japan, Tokyo), and sirtuin-3 (Sirt3) (Cell Signaling Technology Japan).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Comparison

Journal: Cellular & Molecular Biology Letters

Article Title: SGLT2 inhibitor promotes mitochondrial dysfunction and ER-phagy in colorectal cancer cells

doi: 10.1186/s11658-024-00599-1

Figure Lengend Snippet: iSGLT2 impairs energetic state and modulates SIRT3 levels in CRC. Evaluation of ( A ) GSH/GSSG ratio and ( B ) ATP production coupled respiration, ( C ) maximal and ( D ) basal respiration, and ( E ) coupling efficiency assessed by Seahorse analyzer and ( F ) SIRT3 activity, ( G ) content evaluated by ELISA kit, ( H ) mRNA expression and ( I ) immunoblotting analysis with cropped blots in HCT 116 cells treated with 50 µM iSGLT2 for 72 h (iSGLT2). Control cells (Ctr) were maintained in complete culture medium with the corresponding volume of HBSS-10 mM Hepes. M = molecular weight markers, lane 1 = Ctr, lane 2 = iSGLT2. Western blotting is expressed as arbitrary units (AU), mRNA levels are reported as floating bars with a line representing the median ± SD of n = 3 independent experiments. † p < 0.01 vs. Ctr; ‡ p < 0.001 vs. Ctr, by unpaired Student’s t -test

Article Snippet: The following primary antibodies were used for western blotting detection: anti-sodium-glucose cotransporter-2 (SGLT2, 1:500, Abcam, Cambridge, UK), anti-sirtuin 3 (SIRT3, 1:500, Cell Signaling Technology, Danvers, MA, USA), anti-dipeptidyl peptidase-4 (DPP4, 1:500, Abcam, Cambridge, UK) anti-5' AMP-activated protein kinase alpha-1 (AMPK, 1:1000, Invitrogen, Waltham, Ma, USA), anti-phospho-AMPK (1:1000, Invitrogen, Waltham, Ma, USA), anti-C/EBP homologous protein (CHOP, 1:1000, Elabscience Biotechnology Inc., Houston, TX, USA), anti-protein kinase RNA-like ER kinase (PERK, 1:1000, Elabscience Biotechnology Inc., Houston, TX, USA), anti-activating transcription factor 6 (ATF6, 1:1000, Biorbyt, Cambridge, UK), anti-microtubule-associated protein 1 light chain 3B (LC3B, 1:1000, Abcam, Cambridge, UK), anti-actin (1:5000, Abcam, Cambridge, UK), and anti-α-tubulin (1:3000, Elabscience Biotechnology Inc., Houston, TX, USA).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Molecular Weight

Journal: Cellular & Molecular Biology Letters

Article Title: SGLT2 inhibitor promotes mitochondrial dysfunction and ER-phagy in colorectal cancer cells

doi: 10.1186/s11658-024-00599-1

Figure Lengend Snippet: siSIRT3 opposes iSGLT2-dependent cytotoxicity and mitochondrial injury. Detection of ( A , H ) cytotoxicity, ( B , I ) glucose, ( C , J ) lactate ( D , K ) ATP and ( E , L ) NAD + /NADH levels and cytofluorimetric detection and representative fluorescent images of ( F , G and M , N ) mitochondrial ROS in HCT 116 and HT-29 cells treated with 50 µM iSGLT2 for 72 h (iSGLT2) or transfected with nontargeting siRNA control (NT) or SIRT3 siRNA (siSIRT3) before exposure to iSGLT2 (NT + iSGLT2 or siSIRT3 + iSGLT2). Control cells (Ctr) were maintained in complete culture medium with the corresponding volume of HBSS-10 mM Hepes. Scale bars = 100 µm. † p < 0.01 vs. Ctr; ‡ p < 0.001 vs. Ctr; * p < 0.01 vs. NT; ¶ p < 0.001 vs. NT; • p < 0.05 vs. NT + iSGLT2; § p < 0.01 vs. NT + iSGLT2. Significance was determined using one-way ANOVA with Tukey post hoc test

Article Snippet: The following primary antibodies were used for western blotting detection: anti-sodium-glucose cotransporter-2 (SGLT2, 1:500, Abcam, Cambridge, UK), anti-sirtuin 3 (SIRT3, 1:500, Cell Signaling Technology, Danvers, MA, USA), anti-dipeptidyl peptidase-4 (DPP4, 1:500, Abcam, Cambridge, UK) anti-5' AMP-activated protein kinase alpha-1 (AMPK, 1:1000, Invitrogen, Waltham, Ma, USA), anti-phospho-AMPK (1:1000, Invitrogen, Waltham, Ma, USA), anti-C/EBP homologous protein (CHOP, 1:1000, Elabscience Biotechnology Inc., Houston, TX, USA), anti-protein kinase RNA-like ER kinase (PERK, 1:1000, Elabscience Biotechnology Inc., Houston, TX, USA), anti-activating transcription factor 6 (ATF6, 1:1000, Biorbyt, Cambridge, UK), anti-microtubule-associated protein 1 light chain 3B (LC3B, 1:1000, Abcam, Cambridge, UK), anti-actin (1:5000, Abcam, Cambridge, UK), and anti-α-tubulin (1:3000, Elabscience Biotechnology Inc., Houston, TX, USA).

Techniques: Transfection

Journal: Cellular & Molecular Biology Letters

Article Title: SGLT2 inhibitor promotes mitochondrial dysfunction and ER-phagy in colorectal cancer cells

doi: 10.1186/s11658-024-00599-1

Figure Lengend Snippet: siSIRT3 counteracts cell death mechanisms induced by iSGLT2. Representative cytofluorimetric detection and fluorescent images of ( A , B and F , G ) autophagy, ( C , H ) analysis of annexin V-FITC and propidium iodide (PI)-staining and ( D , E and I , J ) ER stress in HCT 116 and HT-29 cells treated with 50 µM iSGLT2 for 72 h (iSGLT2) or transfected with nontargeting siRNA control (NT) or SIRT3 siRNA (siSIRT3) before exposure to iSGLT2 (NT + iSGLT2 or siSIRT3 + iSGLT2). Control cells (Ctr) were maintained in complete culture medium with the corresponding volume of HBSS-10 mM Hepes. Scale bars = 100 µm. † p < 0.01 vs. Ctr; ‡ p < 0.001 vs. Ctr; * p < 0.01 vs. NT; ¶ p < 0.001 vs. NT; • p < 0.05 vs. NT + iSGLT2; § p < 0.01 vs. NT + iSGLT2. Significance was determined using one-way ANOVA with Tukey post hoc test

Article Snippet: The following primary antibodies were used for western blotting detection: anti-sodium-glucose cotransporter-2 (SGLT2, 1:500, Abcam, Cambridge, UK), anti-sirtuin 3 (SIRT3, 1:500, Cell Signaling Technology, Danvers, MA, USA), anti-dipeptidyl peptidase-4 (DPP4, 1:500, Abcam, Cambridge, UK) anti-5' AMP-activated protein kinase alpha-1 (AMPK, 1:1000, Invitrogen, Waltham, Ma, USA), anti-phospho-AMPK (1:1000, Invitrogen, Waltham, Ma, USA), anti-C/EBP homologous protein (CHOP, 1:1000, Elabscience Biotechnology Inc., Houston, TX, USA), anti-protein kinase RNA-like ER kinase (PERK, 1:1000, Elabscience Biotechnology Inc., Houston, TX, USA), anti-activating transcription factor 6 (ATF6, 1:1000, Biorbyt, Cambridge, UK), anti-microtubule-associated protein 1 light chain 3B (LC3B, 1:1000, Abcam, Cambridge, UK), anti-actin (1:5000, Abcam, Cambridge, UK), and anti-α-tubulin (1:3000, Elabscience Biotechnology Inc., Houston, TX, USA).

Techniques: Staining, Transfection

Journal: Cellular & Molecular Biology Letters

Article Title: SGLT2 inhibitor promotes mitochondrial dysfunction and ER-phagy in colorectal cancer cells

doi: 10.1186/s11658-024-00599-1

Figure Lengend Snippet: DPP4 as a target of SGLT2 and SIRT3. A SIRT3 (left) and SGLT2 (as SLC5A2, right) full interaction networks estimated on STRING database ( https://string-db.org/ ) with the following setting parameters: minimum required interaction score: 0.400; max number of interactors to show in the first shell: 10, and none for the second shell. The dotted line suggests the potential interaction between SIRT3 and DPP4. B , C Immunoblotting analysis with cropped blots of DPP4 protein levels in HCT 116 and HT-29 cells treated with 50 µM iSGLT2 for 72 h (iSGLT2) or transfected with nontargeting siRNA control (NT) or SIRT3 siRNA (siSIRT3) before exposure to iSGLT2 (NT + iSGLT2 or siSIRT3 + iSGLT2). Control cells (Ctr) were maintained in complete culture medium with the corresponding volume of HBSS-10 mM Hepes. M: molecular weight markers; lane 1: Ctr; lane 2: iSGLT2; lane 3: NT; lane 4: NT + iSGLT2; lane 5: SIRT3 − ; lane 6: siSIRT3 + iSGLT2. † p < 0.01 vs. Ctr; * p < 0.01 vs. NT; § p < 0.01 vs. NT + iSGLT2. Significance was determined using one-way ANOVA with Tukey post hoc test

Article Snippet: The following primary antibodies were used for western blotting detection: anti-sodium-glucose cotransporter-2 (SGLT2, 1:500, Abcam, Cambridge, UK), anti-sirtuin 3 (SIRT3, 1:500, Cell Signaling Technology, Danvers, MA, USA), anti-dipeptidyl peptidase-4 (DPP4, 1:500, Abcam, Cambridge, UK) anti-5' AMP-activated protein kinase alpha-1 (AMPK, 1:1000, Invitrogen, Waltham, Ma, USA), anti-phospho-AMPK (1:1000, Invitrogen, Waltham, Ma, USA), anti-C/EBP homologous protein (CHOP, 1:1000, Elabscience Biotechnology Inc., Houston, TX, USA), anti-protein kinase RNA-like ER kinase (PERK, 1:1000, Elabscience Biotechnology Inc., Houston, TX, USA), anti-activating transcription factor 6 (ATF6, 1:1000, Biorbyt, Cambridge, UK), anti-microtubule-associated protein 1 light chain 3B (LC3B, 1:1000, Abcam, Cambridge, UK), anti-actin (1:5000, Abcam, Cambridge, UK), and anti-α-tubulin (1:3000, Elabscience Biotechnology Inc., Houston, TX, USA).

Techniques: Western Blot, Transfection, Molecular Weight