Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 2. SGLT2 is upregulated in astrocyte cell bodies and astrocyte endfeet following cerebral ischemia. (a–e) Double immunolabeling for SGLT2 (green) and NeuN (a,b) or GFAP (c–e) (red) in contralateral (Contra) and ipsilateral (Ipsi) brain sections from mice post-MCAo/R (2/6 h); merged images are shown in a, b, d, e; individual labelings are shown in c, d—inset, e—inset; the images shown are representative of findings in 3 mice; scale bars, 25 µm except insert, 10 µm.

Article Snippet: We studied SGLT2 expression using a commercial rabbit polyclonal anti-SGLT2 antibody (#NBP1-92384; Novus Biologicals, Centennial, CO, USA).

Techniques: Immunolabeling

Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 3. Slc5a2 mRNA and SGLT2 protein are upregulated in astrocytes following cerebral ischemia. (a–c) Images (a) and quantification (b,c) of RNAscope for Slc5a2 (red) and Aqp4 (green) mRNA in contralateral (Contra) and ipsilateral (Ipsi) brain sections from mice post-MCAo/R (2/6 h); scale bar, 25 µm; data from 3 mice per group; **, p < 0.01. (d) qPCR for Slc5a2 mRNA in astrocytes and neurons isolated from the contralateral (Contra) and ipsilateral (Ipsi) MCA territory of mice following MCAo/R (2/6 h) and uninjured controls (naïve), expressed as fold-change; 5 mice per group; *, p < 0.05. (e) Quantitative immunohistochemistry for SGLT2 expressed within regions of interest determined by NeuN; data from 7 mice per group; ns, not significant. (f,g) Immunoblot (f) and quantification of immunoblot (g) for SGLT2 in astrocytes isolated from the contralateral (Contra) and ipsilateral (Ipsi) MCA territory of mice following MCAo/R (2/6 h) and uninjured controls (naïve), expressed as fold-change; PC, positive control (kidney lysate); β-actin used as a loading control; 3 mice per group.

Article Snippet: We studied SGLT2 expression using a commercial rabbit polyclonal anti-SGLT2 antibody (#NBP1-92384; Novus Biologicals, Centennial, CO, USA).

Techniques: RNAscope, Isolation, Immunohistochemistry, Western Blot, Positive Control, Control

Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 4. D-glucose-induced Na+ influx in post-ischemic astrocytes is inhibited by canagliflozin and by the astrocyte-specific deletion of Slc5a2/SGLT2. (a) Live cell image of a tdTomato-expressing astrocyte in an ex vivo brain slice from an MCAo/R (2/6 h) mouse, following incubation with ING-2; scale bar, 25 µm. (b–e) Time course of Na+ influx with step change in D-glucose from 2 to 10 mM in ipsilateral (Ipsi) vs. contralateral (Contra) astrocytes (b); in ipsilateral astrocytes without and with pre-incubation with canagliflozin (5 µM) (c); in ipsilateral astrocytes without and with the addition of canagliflozin (5 µM) at the time indicated by the bar (d); in ipsilateral astrocytes from Ast-Slc5a2WT

Article Snippet: We studied SGLT2 expression using a commercial rabbit polyclonal anti-SGLT2 antibody (#NBP1-92384; Novus Biologicals, Centennial, CO, USA).

Techniques: Expressing, Ex Vivo, Slice Preparation, Incubation

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: SIRT6 and SGLT2 expression in atherosclerotic plaques . ( A ) Representative confocal laser-scanning microscope images of SIRT6 and SGLT2 expression levels (red) in deparaffinized atherosclerotic plaques from non-diabetic patients, current SGLT2i users, and never SGLTi users. The von Willebrand factor (vWf, green) was used to properly localize the immunofluorescence signals in endothelial cells, while DAPI staining was used for nuclei counterstaining (blue). Scale Bar = 5 μm. (B) The ImageJ software carried out arbitrary fluorescence units (AFU) of SIRT6 and SGLT2. ∗∗p < 0.01 versus plaque specimen from patients without diabetes, #p < 0.05 never SGLT2i users. Protein expression levels of (C) SIRT6 (D) SGLT2 and (E) NF- B ( NF - κB ) in plaques from diabetic, non-diabetic, and diabetic SGLT2i-user patients. (F) Protein quantification was performed using β-Actin and α-tubulin as the internal control. SGLT2 protein expression detected by using anti-SGLT2 antibody from Cell Signaling Technology. Lane 1 = protein ladder molecular weight markers, lane 2 = non-diabetic, lane 3 = never SGLT2i users, lane 4 = SGLT2i users. The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU) with ∗∗p < 0.01 versus patients without diabetes, #p < 0.05 versus never SGLT2i users.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques: Expressing, Laser-Scanning Microscopy, Immunofluorescence, Staining, Software, Fluorescence, Molecular Weight

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: Atherosclerotic plaque phenotypes . ( A ) Immunochemistry for nitrotyrosine (X40), Tumor Necrosis Factor-alpha (TNF-α) (X40), collagen content (X40), macrophages (CD68) (X40), and Matrix metallopeptidase 9 (MMP-9) (X40), and in non-diabetic, current Sodium-Glucose co-transporter-2 inhibitor (SGLT2i)-user, and never SGLT2i-user asymptomatic plaques. Similar regions of plaque are shown. These results are typical of control, current SGLT2i-user, and never SGLT2i-user asymptomatic plaques. Negative controls were presented in Supplementary Figure 8 . (B) Nitrotyrosine, TNF-α, collagen content, CD68, and MMP-9 in current SGLT2i-user and never SGLT2i-user asymptomatic plaques (The box plots show the median, 25th and 75th percentiles, range, and extreme values). ∗P < 0.05 vs plaques from patients without diabetes. §P < 0.05 vs current SGLT2i-user plaques.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques:

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: Survival from MACE Cox regression analysis (adjusted for age, sex, BMI, blood pressure, heart rate, cholesterol, HDL cholesterol, LDL cholesterol, triglyceride levels, heart disease, hypertension, dyslipidemia, smoking, b-blockers, ACE inhibitors, calcium inhibitors, thiazide diuretics, and aspirin) according to diabetic status (A), SGLT2i therapy (B), and SGLT2 carotid atherosclerotic plaque content (C).

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques:

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: SGLT2 inhibitor restored SIRT6 expression levels during hyperglycemia . (A, B) Representative confocal images of SIRT6 and SGLT2 (red), vimentin (green), and (G) their fluorescence intensity determination, performed by using ImageJ software and expressed as arbitrary fluorescence units. Scale Bar = 10 μm ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc. (C–E) Representative Western blot images and analysis of SIRT6 and SGLT2 expression levels in endothelial cells pre-treated for 8 h with 5 μM SGLT2i before 48 h hGluc (25 mM) stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = vehicle, lane 4 = SGLT2i, lane 5 = hGluc, lane 6 = SGLT2i+hGluc. (F–H) Western blot image and analysis of NF-kB and MMP-9 expression level in EC pre-treated for 8 h with 5 μM SGLT2i before 48 h hGluc (25 mM) stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = vehicle, lane 4 = SGLT2i, lane 5 = hGluc, lane 6 = SGLT2i+hGluc. The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units. α-Tubulin or GAPDH were used as internal control. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques: Expressing, Fluorescence, Software, Western Blot, Molecular Weight

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: SIRT6 mediates both SGLT2 expression and endothelial response against hyperglycemia damages . (A) SIRT6 and (B) SGLT2 expression levels in EC after SIRT6 silencing and/or SGLT2i pre-treatment for 8 h. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = scramble siRNA, lane 4 = SIRT6-siRNA, lane 5 = SGLT2i, lane 6 = SIRT6-siRNA+SGLT2i. α-Tubulin or GAPDH were used as internal control. (C) The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, ∗∗∗p < 0.001 vs Ctr, §p < 0.05 vs SIRT6-siRNA. (D, E) SGLT2 and (F, G) TNF-α protein levels assessed by Western blot analyses. Endothelial cells, after SIRT6 silencing, were subjected or not subjected to 8 h of pre-treatment with SGLT2i before starting 48 h of hGluc stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = scramble siRNA, lane 4 = SIRT6-siRNA, lane 5 = SGLT2i, lane 6 = SIRT6-siRNA+SGLT2i, lane 7 = hGluc, lane 8 = SIRT6-siRNA+hGluc, lane 9 = SGLT2i+hGluc, lane 10 = SIRT6 siRNA+SGLT2i+hGluc. β-Actin or GAPDH were used as the internal control. (H) TNF-α cytokine levels in EC measured after SIRT6 silencing and SGLT2i pre-treatment before starting 48 h of hGluc incubation. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc, §p < 0.05 vs SIRT6 siRNA.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques: Expressing, Molecular Weight, Software, Western Blot, Incubation

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: SIRT6 gene silencing blocks the SGLT2i protective effects against hyperglycemia injury . (A) Cytotoxicity, assessed by LDH Assay Kit-WST, and (B, C) cytokine levels in endothelial cells measured after SIRT6 silencing and hGluc stress condition for 48 h, preceded or not by SGLT2i pre-treatment for 8 h. (D, E) Intracellular ROS content detected by flow cytometry analysis using DCF probe. EC after SIRT6-siRNA were subjected or not subjected to 8 h SGLT2i pre-treatment before starting 48 h of incubation with hGluc. (F) Representative images of confocal laser scanning analyses of mitochondrial ROS generation detected by MitoSOX probe and (G) mitochondrial superoxide levels detected by flow cytometry analysis. Results are expressed as median fluorescence intensity (MFI). Scale bars = 10 μm. The cytoskeleton is marked with Phalloidin 488 (green), while DAPI was used for the nuclei counterstain (blue). (H) Representative confocal images of NF- B ( NF - κB ) (red) and vimentin (green) and (I) fluorescence intensity analysis, performed using ImageJ software, expressed as arbitrary fluorescence units. (J, K) SIRT6 protein levels, assessed by Western blot analysis. Endothelial cells, after SGLT2 silencing, were subjected or not subjected to 8 h of pre-treatment with SGLT2i before starting 48 h of hGluc stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = vehicle, lane 4 = SGLT2i, lane 5 = hGluc, lane 6 = SGLT2i+hGluc, lane 7 = scramble siRNA, lane 8 = SGLT2-siRNA, lane 9 = SGLT2-siRNA+hGluc, lane 10 = SGLT2-siRNA+SGLT2i+hGluc. The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). α-Tubulin was used as an internal control. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques: Lactate Dehydrogenase Assay, Flow Cytometry, Incubation, Fluorescence, Software, Western Blot, Molecular Weight

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: (A–B) Rose-colored SGLT1 fluorescence and pink-colored SGLT2 fluorescence could be seen in the adrenal cross sections of the four groups. (C) Semi-quantitative analysis of SGLT1 and SGLT2 expression in the adrenal glands of four groups showed that SGLT1 and SGLT2 expression levels were higher in mice after stress than in the corresponding non-stressed mice. (D) Expression of SGLT1 and SGLT2 was determined by Western blot analysis. (E) mRNA expression of SGLT1 and SGLT2 was detected by qPCR.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Fluorescence, Expressing, Western Blot

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: CYP11B2 labeled in red showed the adrenal cortex region, while tyrosine hydroxylase (TH) labeled in green showed the adrenal medulla. The nucleus was stained blue by DAPI. SGLT1 was detected by a rose and SGLT2 by a pink. The distribution of SGLT1 and SGLT2 in the adrenal glands of mice in the CON, CON+CS, HF+Apoe -/- , and HF+Apoe -/- +CS groups was observed to be altered by chronic stress.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Labeling, Staining

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: (A) The distribution of SGLT1 was observed under low and high magnification, and the differences in SGLT1 expression level in the adrenal gland of mice in the four groups were analyzed by semi-quantitative methods. (B) The distribution of SGLT2 was observed under high and low magnification in the adrenal gland, and the differences in expression between the four groups were analyzed. (C) Edema, glycogen content, and reticulocyte fiber breakage were positively correlated with the expression levels of SGLT1 and SGLT2 in the adrenal gland, respectively.* P < 0.05, ** P < 0.01, *** P < 0.001; ns =no significance.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Expressing

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: (A) Best training score of 0.0096775 with an epoch of 3000 after BP-neural network training for predicting intima-media thickness of abdominal aorta from SGLT1 and SGLT2 expression levels. (B) Relativity of 0.9841 with good correlation between input and output quantities. (C–D) Validation of the predicted data and the original values, with only small differences. (E) Best training score of 0.012396 with an epoch of 3000 after training the BP-neural network for predicting the internal diameter of abdominal aorta from SGLT1 and SGLT2 expression levels. (F) Relativity of 0.9805, with a good correlation between input and output volumes. (G–H) Validation of predicted data and original values.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Biomarker Discovery

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: (A) The ROC curves showed that the expression level of SGLT1 in the adrenal gland sensitively and specifically predicted adrenal edema, broken reticular fibers, and glycogen content. (B) The ROC curves showed that the expression level of SGLT2 in the adrenal gland sensitively and specifically predicted adrenal edema, broken reticular fibers, and glycogen content.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Expressing

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: (A) The predicted value of SGLT1 and SGLT2 expression levels on adrenal edema was 0.9596 with a mean error of 3.31% by the SVM method. (B) The predicted value of SGLT1/2 expression levels for broken reticular fibers was 0.8246 with a mean error of 1.81%. (C) The predicted value of SGLT1/2 expression levels for glycogen content was 0.9478 with a mean error of 2.15%.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Expressing

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: (A–B) Best training score of 0.018092 at epoch 2999 and relativity of 0.96057 of BP-neural network for predicting adrenal edema from SGLT1 and SGLT2 expression levels after training. (C–D) Validation of the predicted values with the original values revealed only small differences. (E–F) A high-risk warning indicator for adrenal edema derived from SGLT1 and SGLT2 expression levels was found by an interpolation algorithm and presented with a three-dimensional (3D) stereogram of the warning range.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Biomarker Discovery, Derivative Assay

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: (A–B) The optimal training score for SGLT1 and SGLT2 to predict adrenal reticular fiber breakage was 0.01571 at epoch 3000 with relativity of 0.96383. (C–D) The predicted data were verified against the original values and a significant difference was found between the two. (E–F) High-risk warning indicators for adrenal reticular fiber breakage were determined from SGLT1 and SGLT2 expression levels, and the warning range was presented with a 3D stereogram.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Expressing

Journal: PeerJ

Article Title: Adrenal SGLT1 or SGLT2 as predictors of atherosclerosis under chronic stress based on a computer algorithm

doi: 10.7717/peerj.15647

Figure Lengend Snippet: (A–B) The best training score was 0.013784 at epoch 3000 with a relativity of 0.97073. (C–D) Validation revealed only small differences between the predicted values and the original values. (E–F) High-risk warning indicators for adrenal gland glycogen content were determined from SGLT1 and SGLT2 expression levels, and the warning range was presented with a 3D stereogram.

Article Snippet: Transverse sections of adrenal glands were incubated with SGLT1 Polyclonal Antibody (1:200, bs-1128R; BIOSS, Beijing, China) and SGLT2 Polyclonal Antibody (1:200, 24654-1-AP; Proteintech, Rosemont, IL, USA).

Techniques: Biomarker Discovery, Expressing

Journal: Journal of Biological Chemistry

Article Title: Inhibition of soluble epoxide hydrolase alleviates insulin resistance and hypertension via downregulation of SGLT2 in the mouse kidney

doi: 10.1016/j.jbc.2021.100667

Figure Lengend Snippet: Figure 2. HF–HS diet increased soluble epoxide hydrolase (sEH) and sodium–glucose cotransporter 2 (SGLT2) expression in the kidney. A, the expression of sEH and SGLT2 in mice kidneys was evaluated by immunohistochemical staining. The black arrow represents the high expression area of SGLT2. The scale bar represents 200 μm. B, quantitative analysis of immunohistochemical staining of sEH and SGLT2. C and D, representative Western blot and quantitation of sEH and SGLT2 expression. n = 6 mice in each group. Data are shown as mean ± SD. **p < 0.01. HF–HS, high-fat and high- salt; SC–NS, standard chow–normal salt.

Article Snippet: Antibodies against the following proteins were used in this study: sEH (sc-166961; dilution 1:500 for WB, 1:200 for IHC staining), SGLT2 (sc-393350; dilution 1:500 for WB, 1:200 for IHC), CD68 (SC-17832; dilution 1:1000 for WB, 1:500 for IHC), and tumor necrosis factor α (sc-12744; dilution 1:500 for WB, 1:250 for IHC) were purchased from Santa Cruz.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Inhibition of soluble epoxide hydrolase alleviates insulin resistance and hypertension via downregulation of SGLT2 in the mouse kidney

doi: 10.1016/j.jbc.2021.100667

Figure Lengend Snippet: Figure 4. TPPU administration lowered hypertension and improved insulin resistance by inhibiting sodium and glucose reabsorption mediated by 14,15-epoxyeicosatrienoic acid (14,15-EET)–triggered reduced sodium–glucose cotransporter 2 (SGLT2) expression. A, urine volume (n = 9 mice). B, urine sodium excretion (n = 6 mice). C, urine glucose excretion (n = 7 mice). D, representative immunohistochemical staining of SGLT2 in the kidney. The scale bar represents 100 μm. E and F, representative Western blot and quantitation of SGLT2 in mice kidney (n = 6 mice). G, soluble epoxide hydrolase (sEH) activity in kidney tissue (n = 4 mice). H, 14,15-EET levels in kidney tissue (n = 4 mice). Data are shown as mean ± SD. **p < 0.01. EET, epoxyeicosatrienoic acid; HF–HS, high-fat and high-salt; SC–NS, standard chow/normal salt; TPPU, trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea.

Article Snippet: Antibodies against the following proteins were used in this study: sEH (sc-166961; dilution 1:500 for WB, 1:200 for IHC staining), SGLT2 (sc-393350; dilution 1:500 for WB, 1:200 for IHC), CD68 (SC-17832; dilution 1:1000 for WB, 1:500 for IHC), and tumor necrosis factor α (sc-12744; dilution 1:500 for WB, 1:250 for IHC) were purchased from Santa Cruz.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitation Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Inhibition of soluble epoxide hydrolase alleviates insulin resistance and hypertension via downregulation of SGLT2 in the mouse kidney

doi: 10.1016/j.jbc.2021.100667

Figure Lengend Snippet: Figure 6. 14,15-Epoxyeicosatrienoic acid (14,15-EET) prevented the upregulation in sodium–glucose cotransporter 2 (SGLT2) expression in human proximal tubule epithelial cells (HK-2) treated with palmitic acid and NaCl via inhibiting the activation of the inhibitory kappa B kinase α/β (IKKα/ β)/NF-κB signaling pathway. A and B, human renal proximal tubule HK-2 cells were treated with 20 nM NaCl and different concentrations of palmitic acid, the Western blot and quantitation of SGLT2 and soluble epoxide hydrolase (sEH) expression. C, effects of trifluoromethoxyphenyl-3-(1-propionylpiperidin-4- yl) urea (TPPU) and four types of EETs on NaCl and palmitic acid induced elevated SGLT2 expression in HK-2 cells. D, effects of siEPHX2 on NaCl and palmitic acid induced elevated SGLT2 expression in HK-2 cells. E, effects of different concentrations of 14,15-EET on NaCl and palmitic acid induced activation of NF- κB pathway, bovine serum albumin (BSA) (solvent control), and mannitol (osmotic control). F, representative immunofluorescence staining of NF-κB (red) in

Article Snippet: Antibodies against the following proteins were used in this study: sEH (sc-166961; dilution 1:500 for WB, 1:200 for IHC staining), SGLT2 (sc-393350; dilution 1:500 for WB, 1:200 for IHC), CD68 (SC-17832; dilution 1:1000 for WB, 1:500 for IHC), and tumor necrosis factor α (sc-12744; dilution 1:500 for WB, 1:250 for IHC) were purchased from Santa Cruz.

Techniques: Expressing, Activation Assay, Western Blot, Quantitation Assay, Solvent, Control, Staining

Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 2. SGLT2 is upregulated in astrocyte cell bodies and astrocyte endfeet following cerebral ischemia. (a–e) Double immunolabeling for SGLT2 (green) and NeuN (a,b) or GFAP (c–e) (red) in contralateral (Contra) and ipsilateral (Ipsi) brain sections from mice post-MCAo/R (2/6 h); merged images are shown in a, b, d, e; individual labelings are shown in c, d—inset, e—inset; the images shown are representative of findings in 3 mice; scale bars, 25 µm except insert, 10 µm.

Article Snippet: The immunoblot of the kidney lysate showed a pair of bands, ~60 and ~65 kDa, with a third band, ~26 kDa, which were absent or minimal in lung lysate and were blocked by pre-absorption of the anti-SGLT2 antibody with the recombinant SGLT2 antigen peptide (#NBP1-92384PEP; Novus Biologicals) (Figure S3a).

Techniques: Immunolabeling

Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 3. Slc5a2 mRNA and SGLT2 protein are upregulated in astrocytes following cerebral ischemia. (a–c) Images (a) and quantification (b,c) of RNAscope for Slc5a2 (red) and Aqp4 (green) mRNA in contralateral (Contra) and ipsilateral (Ipsi) brain sections from mice post-MCAo/R (2/6 h); scale bar, 25 µm; data from 3 mice per group; **, p < 0.01. (d) qPCR for Slc5a2 mRNA in astrocytes and neurons isolated from the contralateral (Contra) and ipsilateral (Ipsi) MCA territory of mice following MCAo/R (2/6 h) and uninjured controls (naïve), expressed as fold-change; 5 mice per group; *, p < 0.05. (e) Quantitative immunohistochemistry for SGLT2 expressed within regions of interest determined by NeuN; data from 7 mice per group; ns, not significant. (f,g) Immunoblot (f) and quantification of immunoblot (g) for SGLT2 in astrocytes isolated from the contralateral (Contra) and ipsilateral (Ipsi) MCA territory of mice following MCAo/R (2/6 h) and uninjured controls (naïve), expressed as fold-change; PC, positive control (kidney lysate); β-actin used as a loading control; 3 mice per group.

Article Snippet: The immunoblot of the kidney lysate showed a pair of bands, ~60 and ~65 kDa, with a third band, ~26 kDa, which were absent or minimal in lung lysate and were blocked by pre-absorption of the anti-SGLT2 antibody with the recombinant SGLT2 antigen peptide (#NBP1-92384PEP; Novus Biologicals) (Figure S3a).

Techniques: RNAscope, Isolation, Immunohistochemistry, Western Blot, Positive Control, Control

Journal: Cells

Article Title: Canagliflozin, an Inhibitor of the Na + -Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

doi: 10.3390/cells12182221

Figure Lengend Snippet: Figure 4. D-glucose-induced Na+ influx in post-ischemic astrocytes is inhibited by canagliflozin and by the astrocyte-specific deletion of Slc5a2/SGLT2. (a) Live cell image of a tdTomato-expressing astrocyte in an ex vivo brain slice from an MCAo/R (2/6 h) mouse, following incubation with ING-2; scale bar, 25 µm. (b–e) Time course of Na+ influx with step change in D-glucose from 2 to 10 mM in ipsilateral (Ipsi) vs. contralateral (Contra) astrocytes (b); in ipsilateral astrocytes without and with pre-incubation with canagliflozin (5 µM) (c); in ipsilateral astrocytes without and with the addition of canagliflozin (5 µM) at the time indicated by the bar (d); in ipsilateral astrocytes from Ast-Slc5a2WT

Article Snippet: The immunoblot of the kidney lysate showed a pair of bands, ~60 and ~65 kDa, with a third band, ~26 kDa, which were absent or minimal in lung lysate and were blocked by pre-absorption of the anti-SGLT2 antibody with the recombinant SGLT2 antigen peptide (#NBP1-92384PEP; Novus Biologicals) (Figure S3a).

Techniques: Expressing, Ex Vivo, Slice Preparation, Incubation