Journal: Journal of Orthopaedic Translation

Article Title: The senolytic agent ABT263 ameliorates osteoporosis caused by active vitamin D insufficiency through selective clearance of senescent skeletal cells

doi: 10.1016/j.jot.2024.08.012

Figure Lengend Snippet: ABT263 intervention corrects oxidative stress and DNA damage caused by 1,25(OH) 2 D insufficiency Lumbar vertebrae of WT, Cyp27b1 +/− , and Cyp27b1 +/− + ABT263 mice. (A & B) Representative micrographs of SOD2 immunohistochemical staining and the percentage of SOD2-positive cells. (C & D) Representative micrographs of γ-H2A.X immunohistochemical staining and the percentage of γ-H2A.X-positive cells. (E & F) Western blot results of SOD2 and γ-H2A.X proteins. Values are mean ± S.E.M. of 6 determinations per group. ∗∗∗: P < 0.001, compared to WT mice. ##: P < 0.01; ###: P < 0.001, compared to Cyp27b1 +/− mice.

Article Snippet: Subsequently, the membranes were probed with specific primary antibodies against the following proteins: BCL2 (Abcam), Bax (Santa Cruz), Runx2 (Santa Cruz), Osteocalcin (Santa Cruz), SOD2 (NB100-1992, Novus Biologicals), γ-H2A.X (#80312S, Cell Signaling Technology), p16 (Santa Cruz), TNFα (Santa Cruz), and β-actin (Cell Signaling Technology).

Techniques: Immunohistochemical staining, Staining, Western Blot

Journal: Cancer Research

Article Title: MicroRNA-21 Modulates the Levels of Reactive Oxygen Species by Targeting SOD3 and TNFα

doi: 10.1158/0008-5472.can-12-0639

Figure Lengend Snippet: Figure 3. miR-21 directly targets TNFa, an SOD2 inducer. A, TNFa levels were measured by Western blot analysis in cells with or without stable miR-21 overexpression at different times after 0.5 Gy low-LET ionizing radiation. b-Actin was used as an internal loading control. B, potential miR-21 binding site in the 30-UTR of TNFa and the effects of the binding site on the luciferase activity. Luciferase activity was assayed 48 hours after transfection with the miR- 21 mimic (miR-21) or control RNA (mock). , P < 0.01. C, effects of TNFa on SOD2 expression. Cells overexpressing miR-21 or vector- transfected cells were transiently transfected with the TNFa cDNA (without 30-UTR) or siRNA. At 48 hours after transfection, the cells were collected and analyzed by Western blot analysis. b-Actin was used as an internal loading control. D, ROS levels (left, O2 ; right, H2O2) were measured using the cells (as described in C), with up- or downregulated TNFa at 3 hours after 0.5 Gy low-LET IR. The data are an average of 3 separate experiments. , P < 0.01.

Article Snippet: The siRNA against SOD2, SOD3, TNFa, the control RNA and the antibody against SOD1, SOD2, or b-actin was purchased from Santa Cruz Biotech Inc.

Techniques: Western Blot, Over Expression, Control, Binding Assay, Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation

Journal: Cancer Research

Article Title: MicroRNA-21 Modulates the Levels of Reactive Oxygen Species by Targeting SOD3 and TNFα

doi: 10.1158/0008-5472.can-12-0639

Figure Lengend Snippet: Figure 4. miR-21 increases IR-induced cell transformation partially via targeting SOD3 and TNFa. A, the data reflect the number of colonies that were grown from cells with or without miR-21 overexpression in soft agar for 3 weeks. The results were obtained from 2 separate experiments with triple dishes per sample in each experiment. , P < 0.01; , P < 0.001. B, similar experiments were carried out as described in A, but with the cells upregulated either by SOD3 or TNFa cDNAs. The results were confirmed in 2 separate experiments with triple dishes per sample in each experiment. , P < 0.001. C, a model proposed to explain how miR-21 could stimulate cell transformation partially through increasing superoxide, thereby contributing to the role of miR-21 in carcinogenesis. miR-21–increased IR-induced superoxide levels through targeting SOD3 and TNFa (a transcriptional activator of SOD2), which contributes to IR- induced cell transformation and carcinogenesis.

Article Snippet: The siRNA against SOD2, SOD3, TNFa, the control RNA and the antibody against SOD1, SOD2, or b-actin was purchased from Santa Cruz Biotech Inc.

Techniques: Transformation Assay, Over Expression

Journal: Biomedicines

Article Title: Aldosterone Excess Induced Mitochondria Decrease and Dysfunction via Mineralocorticoid Receptor and Oxidative Stress In Vitro and In Vivo

doi: 10.3390/biomedicines9080946

Figure Lengend Snippet: Aldosterone suppressed mitochondrial DNA and protein in a dose-dependent manner. ( A ) The effects of different dosages of aldosterone on H9c2 cells. H9c2 cells were treated with different concentrations of aldosterone (10 −10 , 10 −9 , 10 −8 and 10 −7 M) and vehicle (equal volume of DMSO) for 72 h. The mitochondrial DNA copy number was quantified by qPCR. ( B ) The effects of different durations of aldosterone treatment on H9c2 cells. H9c2 cells were treated with 10 −7 M aldosterone and the mitochondrial copy number was quantified by qPCR at 0, 8, 24, 48 and 72 h. ( C ) The effects of different durations of aldosterone treatment on SOD2. H9c2 cells were treated with 10 −7 M aldosterone, and the expression of SOD2 was determined by ELISA. ( D ) The dose effect of aldosterone on mitochondrial COX IV protein. H9c2 cells were treated with different concentrations of aldosterone (vehicle, 10 −10 , 10 −9 , 10 −8 and 10 −7 M) for 72 h. COX IV was stained with anti-COX IV antibodies. The fluorescence intensity of COX IV was measured using a fluorescence microscope. ( E ) H9c2 cells were treated with different concentrations of aldosterone (10 −10 , 10 −9 , 10 −8 and 10 −7 M) for 72 h. COX IV was stained with anti-COX IV antibodies (red), and nuclear DNA was stained with DAPI (blue). The representative images were captured using a fluorescence microscope; magnification ×400. ( F ) The effects of aldosterone on mitochondria (COX IV) and cytosolic (α-Tubulin and GAPDH) protein in H9c2 cells. H9c2 cells were treated with different concentrations of aldosterone (vehicle, 10 −10 , 10 −9 , 10 −8 and 10 −7 M) for 72 h. COX IV, α–Tubulin and GAPDH were determined using Western blot analysis. # p < 0.05 and * p < 0.01, compared between the two groups indicated by the line underneath.

Article Snippet: The superoxide dismutase 2 (SOD2) level in cell lysates was determined using a SOD2 DuoSet IC ELISA kit (DYC3419–2; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Microscopy, Western Blot

Journal: Biomedicines

Article Title: Aldosterone Excess Induced Mitochondria Decrease and Dysfunction via Mineralocorticoid Receptor and Oxidative Stress In Vitro and In Vivo

doi: 10.3390/biomedicines9080946

Figure Lengend Snippet: Aldosterone-associated reduction in mitochondrial DNA and protein via MR and MAPK/P38 pathway. ( A , B ) Role of MRs and glucocorticoid receptors on mitochondrial DNA and SOD2 protein. H9c2 cells were treated with 10 −7 M eplerenone (an MR antagonist), 10 −7 M mifepristone (a glucocorticoid receptor antagonist) and vehicle (equal volume of DMSO) for 1 h prior to 10 −7 M aldosterone treatment. After 72 h, mitochondrial DNA was determined by qPCR and the expression of SOD2 was determined by ELISA. ( C , D ) Role of signaling mediators on mitochondrial DNA and SOD2. H9c2 cells were treated with 5 µg/mL SB203580 (an MAPK/p38 inhibitor), 50 µg/mL PD98059 (an MEK/ERK inhibitor), 50 µg/mL LY294002 (a PI3K/ AKT inhibitor) or vehicle (equal volume of DMSO) for 1 h prior to 10 −7 M aldosterone treatment. After 72 h, mitochondrial DNA was determined by qPCR and the expression of SOD2 was determined by ELISA. * p < 0.01, compared between the two groups indicated by the line underneath.

Article Snippet: The superoxide dismutase 2 (SOD2) level in cell lysates was determined using a SOD2 DuoSet IC ELISA kit (DYC3419–2; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Aldosterone Excess Induced Mitochondria Decrease and Dysfunction via Mineralocorticoid Receptor and Oxidative Stress In Vitro and In Vivo

doi: 10.3390/biomedicines9080946

Figure Lengend Snippet: Schematic of the signaling of aldosterone-induced cardiac mitochondrial dysfunction in H9c2 cells. Aldosterone-induced cardiac mitochondrial dysfunction through MR/MAPK/p38 and ROS pathways. Mitochondrial DNA, SOD2, COX IV protein and ATP production were suppressed.

Article Snippet: The superoxide dismutase 2 (SOD2) level in cell lysates was determined using a SOD2 DuoSet IC ELISA kit (DYC3419–2; R&D Systems, Minneapolis, MN, USA).

Techniques:

Journal: PloS one

Article Title: Short term exercise induces PGC-1α, ameliorates inflammation and increases mitochondrial membrane proteins but fails to increase respiratory enzymes in aging diabetic hearts.

doi: 10.1371/journal.pone.0070248

Figure Lengend Snippet: Figure 7. Moderate intensity exercise in aged diabetic hearts induces mitochondrial oxidative stress in db/db hearts. (a) Representative micrographs of SOD2 immunofluorescence from heart sections. Please note the red punctate staining for SOD2 for all groups except db/db+Exe hearts. Magnification: 2006. (b) Quantification of SOD2 immunopositivity in heart sections as a percentage of total cardiac surface area. (c) TBARS levels in the heart as determined by biochemical assays. Data was analyzed using two-way ANOVA with Bonferroni tests, p,0.05 (n = 6). *P,0.05 versus corresponding Wt group; #P,0.05 versus corresponding sedentary group. Abbreviations: SOD2, mitochondrial isoform of superoxide dismutase; TBARS, thiobarbituric acid reactive substances. doi:10.1371/journal.pone.0070248.g007

Article Snippet: Rabbit polyclonal antibodies directed against F4/80 (Cedarlane), SOD2 (Santa Cruz Biotechnology), VDAC-1 and CoxIV (ABMGood, Richmond, CA) were used as primary antibodies with anti-rabbit Dylight594 or 488 (ABMGood) as secondary antibodies.

Techniques: Immunofluorescence, Staining

Journal: Cell death & disease

Article Title: Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress.

doi: 10.1038/s41419-021-04486-x

Figure Lengend Snippet: Fig. 1 SOD2high/MCTS1high expression is a new poor prognostic marker in invasive breast carcinoma. A, B The relapse-free survival (RFS) of breast cancer patients classified by low (n = 2947) and high (n = 1004) MCTS1 expression (A) or by low (n = 1449) and high (n = 2502) SOD2 expression (B) was evaluated using Kaplan–Meier (KM) Plotter analysis. C The correlation of MCTS1 and SOD2 gene expression in breast cancer patients (n = 159) was analyzed using the Pawitan dataset in the Oncomine cancer profiling database. D–F SOD2 expression was analyzed in TissueScan cDNA arrays with normal breast tissue (n = 11) and breast tumor biopsies (n = 124) across distinctive tumor stages (D, E), molecular subtypes and TNM classifications (F). The SOD2 mRNA levels in tumor samples were normalized to internal ACTB (β-actin) mRNA levels and then compared with the levels in normal breast samples. Data are presented as the mean ± s.e.m. G KM Plotter was used to estimate the survival of patients with MCTS1high/SOD2high (n = 37) expression compared with that of those with MCTS1low/SOD2low (n = 60) expression using the Pawitan dataset in the Oncomine database. H, I RFS was estimated for ER-negative (n = 434) (H) and basal-like (n = 309) (I) breast cancer patients with MCTS1high/SOD2high or MCTS1low/SOD2low expression using KM Plotter. Statistical analysis was performed using the log-rank Mantel–Cox test (A, B, and G–I), Pearson product-moment correlation coefficient (C), Kruskal–Wallis test followed by Dunn’s multiple comparison test (D) and χ2 test (E, F).

Article Snippet: To generate cells overexpressing MnSOD, True-ORF pCMV6-Entry-Flag-tagged SOD2 (#RC202330) and Sod2 (#MR202568) (all from OriGene) were transfected into MDA-MB-231 (IV2-3) and 4T1 cells with Lipofectamine 3000 (Thermo Fisher Scientific), and the transfectants were selected with 100 or 200 μg/ ml neomycin (G418) for 2 weeks.

Techniques: Expressing, Marker, Gene Expression, Comparison

Journal: Cell death & disease

Article Title: Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress.

doi: 10.1038/s41419-021-04486-x

Figure Lengend Snippet: Fig. 2 Overexpression of MCT-1 stabilizes Nrf2 and induces MnSOD. A MnSOD, Nrf2, and catalase levels were examined in normal MCF-10A cells and metastatic MDA-MB-231 cells. B The SOD2 mRNA levels in MCF-10A, MDA-MB-231, and HCC1395 cells without (control) or with MCT- 1 overexpression were measured by qRT-PCR. C, D MnSOD, Nrf2 and catalase levels were assayed upon MCT-1 gene silencing (shMCT-1 #1, #2 or #3) in MCF-10A (C) and MDA-MB-231 (D) cells. E Upon cycloheximide (CHX) treatment for the indicated times, Nrf2 protein stability and its amount relative to that of internal ACTB (right) were compared in MDA-MB-231 cells (control vs. MCT-1). Representative images (left) from four independent experiments are shown. F, G NFE2L2 mRNA levels were analyzed across distinctive breast tumor stages (F) and molecular subtypes or TNM classifications (G) using TissueScan breast cancer cDNA arrays. The NFE2L2 mRNA levels in tumor samples were normalized to the internal ACTB mRNA level and then compared with the levels in normal breast samples. H-I KM Plotter analysis estimated relapse-free survival in basal-like (H) breast cancer patients with MCTS1high/NFE2L2high (n = 155) expression versus those with MCTS1low/NFE2L2low (n = 154) expression and in breast cancer patients with lymph node metastasis (LN positive) (I) stratified by a SOD2high/NFE2L2high (n = 283) or SOD2low/ NFE2L2low (n = 283) signature. J The correlation of SOD2 and NFE2L2 expression in TNBC patients was analyzed using the Stickeler dataset (n = 32) in the Oncomine database. Data (B, E) are presented as the mean ± s.e.m. Statistical analysis was performed using a two-tailed unpaired Student’s t-test (B), linear regression followed by an analysis of variance (ANOVA) (E), the χ2 test (F, G), the log-rank Mantel–Cox test (H, I), and the Pearson product-moment correlation coefficient (J). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: To generate cells overexpressing MnSOD, True-ORF pCMV6-Entry-Flag-tagged SOD2 (#RC202330) and Sod2 (#MR202568) (all from OriGene) were transfected into MDA-MB-231 (IV2-3) and 4T1 cells with Lipofectamine 3000 (Thermo Fisher Scientific), and the transfectants were selected with 100 or 200 μg/ ml neomycin (G418) for 2 weeks.

Techniques: Over Expression, Control, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Cell death & disease

Article Title: Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress.

doi: 10.1038/s41419-021-04486-x

Figure Lengend Snippet: Fig. 4 The MCT-1/IL-6/MnSOD axis induces mROS generation and M2 macrophage polarization. A The amounts of Nrf2 and MnSOD in MDA-MB-231 cells (control vs. MCT-1) were examined upon IL-6 (50 ng/ml) stimulation for different intervals. B IL-6 knockdown (shIL-6) was confirmed in MDA-MB-231 cells (control vs. MCT-1). C The expression of M2 markers (CD163 and MRC1) were examined after THP-1 macrophages were cocultured with MDA-MB-231 cells (control vs. MCT-1) with or without shIL-6. D Secreted IL-6 levels were measured to evaluate MDA-MB-231 cells with or without MnSOD knockdown (scramble vs. shMnSOD) and MCT-1 overexpression (control vs. MCT-1). E The correlation of SOD2 and IL6 expression in TNBC patients (n = 151) was analyzed using the Gluck dataset in the Oncomine. F The correlation of SOD2 and IL6 expression in TNBC patients (n = 29) was examined using TissueScan breast cancer cDNA arrays. G, H Nrf2, MnSOD, and IL-6 levels were assayed after exposure to DPI (G) or Rotenone (Rot) (H). I Mitochondrial ROS (mROS) levels were quantified in MDA-MB-231 cells (scramble vs. shMCT-1) with or without MnSOD overexpression (MOCK vs. MnSOD) using superoxide fluorescent probe MitoSOX. J mROS levels were quantified in MDA-MB-231 cells (control vs. MCT-1) with or without MnSOD silencing (scramble vs. shMnSOD #7 and #8). K, L The peroxidase (K) and MnSOD dismutase activities (L) of MDA-MB-231 cells with or without MCT-1 knockdown (scramble vs. shMCT-1) and MnSOD overexpression (MOCK vs. MnSOD). M H2O2 release from MDA-MB-231 cells with or without MCT-1 knockdown (scramble vs. shMCT- 1) and MnSOD overexpression (MOCK vs. MnSOD) was measured using Amplex Red. N Quantification of H2O2 released by MDA-MB-231 cells (scramble vs. shMCT-1) in MOCK vs. MnSOD expression conditions upon IL-6 stimulation for 24 h. Data are presented as the mean ± s.e.m. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey–Kramer (C, D, I–K, M-N) or Newman–Keuls (L) post hoc analysis or the Pearson correlation coefficient (E, F). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: To generate cells overexpressing MnSOD, True-ORF pCMV6-Entry-Flag-tagged SOD2 (#RC202330) and Sod2 (#MR202568) (all from OriGene) were transfected into MDA-MB-231 (IV2-3) and 4T1 cells with Lipofectamine 3000 (Thermo Fisher Scientific), and the transfectants were selected with 100 or 200 μg/ ml neomycin (G418) for 2 weeks.

Techniques: Control, Knockdown, Expressing, Over Expression

Journal: Cell death & disease

Article Title: Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress.

doi: 10.1038/s41419-021-04486-x

Figure Lengend Snippet: Fig. 6 Targeting MnSOD suppresses TNBC progression and M2 macrophage infiltration. A MDA-MB-231 cells (1 × 106) with or without MCT-1 overexpression (control vs. MCT-1) and MnSOD knockdown (scramble vs. shMnSOD) were orthotopically implanted into the mammary fat pad of nude mice (n = 6 ~ 9). Tumor growth rates were monitored weekly. B Representative tumor images (left) and tumor burdens (righ) were assessed at week 9 in xenograft mice. C Tumor mortality was analyzed after orthotopic injection of the indicated MDA-MB-231 cells into nude mice (n = 8). D Immunohistochemistry was used to examine tumor-associated CD163(+) M2 macrophages (denoted by red arrowheads). Scale bar, 50 µm. E Flow cytometric analysis of M2 macrophages (CD206(+) in F4/80(+) populations) residing in tumors was performed. F The correlation of the SOD2 expression in invasive breast carcinoma patients of basal-like subtypes (n = 191) in The Cancer Genome Atlas (TCGA) cohort with the infiltration of M2 macrophages was evaluated using TIMER 2.0 and analyzed by the CIBERSORT-ABS tool. Expression levels are indicated as log2 transcripts per million (TPM). G Schematic figure depicting that amplification of the IL-6/MCT-1/Nrf2/MnSOD signaling loop aggravates TNBC progression/stemness and enhances M2 functions. MnSOD silencing thereby abolishes TNBC growth and improves M1 macrophage-mediated phagocytosis. Data are presented as the mean ± s.e.m. Statistical analysis was performed using a two-tailed unpaired Student’s t-test (A, D), the log-rank Mantel–Cox test (C), two-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc analysis (B, E), and the Spearman’s rank correlation coefficient (Rho) (F). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: To generate cells overexpressing MnSOD, True-ORF pCMV6-Entry-Flag-tagged SOD2 (#RC202330) and Sod2 (#MR202568) (all from OriGene) were transfected into MDA-MB-231 (IV2-3) and 4T1 cells with Lipofectamine 3000 (Thermo Fisher Scientific), and the transfectants were selected with 100 or 200 μg/ ml neomycin (G418) for 2 weeks.

Techniques: Over Expression, Control, Knockdown, Injection, Immunohistochemistry, Expressing, Two Tailed Test

Journal: BMC pharmacology & toxicology

Article Title: Effect of luteolin on oxidative stress and inflammation in the human osteoblast cell line hFOB1.19 in an inflammatory microenvironment.

doi: 10.1186/s40360-024-00764-4

Figure Lengend Snippet: Fig. 1 Effect of LPS on the protein expression of CRP, IL-2, and SOD in hFOB1.19 cells, as determined by ELISA. ELISA analysis demonstrated that after treat ment with LPS, the concentrations of CRP and IL-2 gradually increased in hFOB1.19 cells and then decreased after 24 h of treatment (A and B). SOD activity gradually decreased in hFOB1.19 cells and then increased after 24 h of treatment (C). The data are expressed as the means ± SDs (**p < 0.01, *p < 0.05)

Article Snippet: The concentrations of TNF-α, IL-8, IL-6 and SOD were monitored by using TNF-α ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA; Cat No: DTA00D), IL-8 ELISA kits (R&D Systems, Cat No: D8000C), IL-6 ELISA kits (R&D Systems, Cat No: D6050B), and SOD ELISA kits (R&D Systems, Cat No: DYC3419-2), respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Nucleic Acids Research

Article Title: MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

doi: 10.1093/nar/gkq718

Figure Lengend Snippet: Proteins differentially expressed in HK2 cells in response to TGFβ1

Article Snippet: Superoxide dismutase 2 (SOD2) human cDNA ORF clone was obtained from OriGene.

Techniques: Membrane

Journal: Nucleic Acids Research

Article Title: MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

doi: 10.1093/nar/gkq718

Figure Lengend Snippet: Verification of proteomic results. HK2 cells were treated as described in . Protein samples used for the western blot analysis were independent of those used for the proteomic analysis. TPM1, tropomyosin 1; SOD2, superoxide dismutase 2. The gel shown for TPM1 was a time course analysis. Only the 48-h time point was plotted in the bar graph for comparison with the proteomic data. n = 4 for proteomics, n = 3 for western blot; *significantly different from vehicle ( P < 0.05 for western blot; see ‘Materials and Methods’ section for the criteria of differential expression for the proteomic analysis).

Article Snippet: Superoxide dismutase 2 (SOD2) human cDNA ORF clone was obtained from OriGene.

Techniques: Western Blot, Comparison, Quantitative Proteomics

Journal: Nucleic Acids Research

Article Title: MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

doi: 10.1093/nar/gkq718

Figure Lengend Snippet: MiRNA-target pairs supported by sequence characteristics and reciprocal expression in human renal epithelial cells treated with TGFβ1

Article Snippet: Superoxide dismutase 2 (SOD2) human cDNA ORF clone was obtained from OriGene.

Techniques: Sequencing, Expressing

Journal: Nucleic Acids Research

Article Title: MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

doi: 10.1093/nar/gkq718

Figure Lengend Snippet: miR-382 targeted SOD2 and contributed to TGFβ1-induced loss of epithelial characteristics in human renal epithelial cells. ( A ) TGFβ1 induced loss of epithelial characteristics in HK2 cells as indicated by typical changes in cell morphology and a suppression of E-cadherin mRNA, an epithelial marker. TGFβ1 was used at 3 ng/ml for 48 h. N = 6, * P < 0.05. ( B ) Knockdown of miR-382. HK2 cells were treated with vehicle or TGFβ1 in the presence of LNA anti-miR-382 or LNA scrambled control anti-miR (100 nM). n = 6, * P < 0.05 vs. no treatment; # P < 0.05 versus TGFβ1 plus control anti-miR. ( C ) Knockdown of miR-382 attenuated the suppression of E-cadherin mRNA by TGFβ1. n = 6–9, * P < 0.05 versus control anti-miR. ( D) miR-382 interacted with the 3′-UTR of SOD2 in Hela cells. Hela cells were transfected with luciferase reporter constructs containing a 3′-UTR segment of SOD2 or a mutated (SOD2-mut) or partially deleted (SOD2-del) segment. The mutations and deletion were described in the text. The effect of anti-miR-382 (100 nM) compared to control, scramble anti-miR was shown. n = 4, * P < 0.05 versus control anti-miR. ( E) miR-382 interacted with 3′-UTR of SOD2 in HK2 cells. HK2 cells were transfected with luciferase reporter constructs as described above. The effect of pre-miR-382 (100 nM), a miR-382 mimic, compared with control, scramble pre-miR was shown. n = 4, * P < 0.05 versus control pre-miR. ( F ) SOD2 expression was suppressed by TGFβ1, but partially restored by knockdown of miR-382. miR-382 in TGFβ1-treated HK2 cells was knocked down with LNA anti-miR (see B). n = 6, * P < 0.05 vs. control anti-miR.

Article Snippet: Superoxide dismutase 2 (SOD2) human cDNA ORF clone was obtained from OriGene.

Techniques: Marker, Knockdown, Control, Transfection, Luciferase, Construct, Expressing

Journal: Nucleic Acids Research

Article Title: MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

doi: 10.1093/nar/gkq718

Figure Lengend Snippet: 3′-UTR reporter constructs

Article Snippet: Superoxide dismutase 2 (SOD2) human cDNA ORF clone was obtained from OriGene.

Techniques:

Journal: Nucleic Acids Research

Article Title: MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

doi: 10.1093/nar/gkq718

Figure Lengend Snippet: SOD2 was protective against TGFβ1-induced loss of epithelial characteristics. ( A ) Overexpression of SOD2 with a Myc-DDK-tagged plasmid (pSOD2). HK2 cells were not transfected (NT) or were transfected with pSOD2 or an empty vector (2 µg for each 3.5-cm dish). Western blot was performed 24 h later using an SOD2 antibody. ( B ) Overexpression of SOD2 attenuated TGFβ1-induced downregulation of E-cadherin. n = 5, * P < 0.05.

Article Snippet: Superoxide dismutase 2 (SOD2) human cDNA ORF clone was obtained from OriGene.

Techniques: Over Expression, Plasmid Preparation, Transfection, Western Blot