Journal: Renal Failure

Article Title: High-glucose induced toxicity in HK-2 cells can be alleviated by inhibition of miRNA-320c

doi: 10.1080/0886022X.2022.2106874

Figure Lengend Snippet: PTEN was the direct target gene of miR-320c. (A) The putative miR-320c binding sites in the PTEN sequence. (B) MiR-320c mimic or mimic NC and pSI-Check2-ATG7-3’UTR plasmid was co-transfected into HEK-293T cells. The relative firefly luciferase activity was determined after transfection for 48 h. (C) miR-320c inhibitor or inhibitor NC were transfected into HK-2 cells and the protein expression of PTEN was conducted by WB and quantified by ImageJ software, with β-actin as a loading control. Bars represent the mean ± SD from three independent experiments. * p < 0.05 and *** p < 0.0001.

Article Snippet: First, to determine the optimal time for HG treatment, HK-2 cells were cultured in a serum-free cell culture medium (Invitrogen, USA) containing 45 mM glucose (HG, Sigma-Aldrich, USA) or 5.5 mM glucose (normal glucose, control), which had a purity ≥ 99.5% and was suitable for the culturing of mammalian cells for 6 h, 12 h, 24 h, and 48 h. Then, for the study, HK-2 cells were treated with HG for 24 h and transfected with small interfering RNA targeting PTEN (si-PTEN), miR-320c inhibitor and its negative control, inhibitor NC (Ribobio, China) at a final concentration of 50-100 nM.

Techniques: Binding Assay, Sequencing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Expressing, Software, Control

Journal: Renal Failure

Article Title: High-glucose induced toxicity in HK-2 cells can be alleviated by inhibition of miRNA-320c

doi: 10.1080/0886022X.2022.2106874

Figure Lengend Snippet: Down-regulation of PTEN reversed the function of miR-320c on HG-induced HK-2 cells. HK-2 cells were transfected with miR-320c inhibitor, si-PTEN, or both and treated with HG, and the expressions of (A) PTEN, p-PI3K, PI3K, AKT, p-AKT (D) E-cadherin, vimentin, and α-SMA were analyzed by WB. (B) PTEN level, (C)p-AKT/AKT and p-PI3K/PI3K ratio, and € E-cadherin, vimentin, and α-SMA were quantified by ImageJ software. Β-actin served as control. (F) Transwell invasion assay was performed, scale bar = 50 μm and (H) numbers of invaded cells were quantified. (O) The migration ability of HK-2 cells was measured by scratch wound assay, and (G) migration ratio was quantified by the migration rate in each group. Scale bar = 100 μm. Bars represent the mean ± SD from three independent experiments. Compared with the control group, *** p < 0.001; compared with the HG group, & p < 0.05, && p < 0.01, &&& p < 0.001, compared with the HG + miR-320c inhibitor, # p < 0.05, ## p < 0.01.

Article Snippet: First, to determine the optimal time for HG treatment, HK-2 cells were cultured in a serum-free cell culture medium (Invitrogen, USA) containing 45 mM glucose (HG, Sigma-Aldrich, USA) or 5.5 mM glucose (normal glucose, control), which had a purity ≥ 99.5% and was suitable for the culturing of mammalian cells for 6 h, 12 h, 24 h, and 48 h. Then, for the study, HK-2 cells were treated with HG for 24 h and transfected with small interfering RNA targeting PTEN (si-PTEN), miR-320c inhibitor and its negative control, inhibitor NC (Ribobio, China) at a final concentration of 50-100 nM.

Techniques: Transfection, Software, Control, Transwell Invasion Assay, Migration, Scratch Wound Assay Assay

Journal: Life

Article Title: Spermine Ameliorates DSS-Induced Ulcerative Colitis in Mice by Improving Mitophagy and Intestinal Microbiota

doi: 10.3390/life16030417

Figure Lengend Snippet: Spermine treatment inhibited NLRP3-mediated inflammatory response in DSS-treated mice. ( A – E ) Genes expressions related to NLRP3-mediated inflammatory response in the colon were measured. ( F – I ) The levels of pro-inflammatory cytokines in the serum. ( J ) Detection of NLRP3 inflammasome protein expression levels in the colon using immunohistochemistry. Data are presented as mean ± SEM (* p < 0.05, *** p < 0.001 vs. control group. ## p < 0.01, and ### p < 0.001 vs. DSS group).

Article Snippet: The sections were then incubated at 4 °C overnight with primary antibodies targeting NLRP3 (1:500, Proteintech, 30109-1-AP, Chicago, IL, USA), PINK1 (1:500, Proteintech, 23274-1-AP, Chicago, IL, USA), and LC3 (1:500, Proteintech, 14600-1-AP, Chicago, IL, USA).

Techniques: Expressing, Immunohistochemistry, Control

Journal: Life

Article Title: Spermine Ameliorates DSS-Induced Ulcerative Colitis in Mice by Improving Mitophagy and Intestinal Microbiota

doi: 10.3390/life16030417

Figure Lengend Snippet: Potential mechanism of Spermine treatment alleviating DSS-induced colitis. First, spermidine enhances mitochondrial autophagy by upregulating the expression of PINK1/Parkin, thereby eliminating damaged mitochondria and reducing the production of reactive oxygen species. This, in turn, inhibits the activation of the NLRP3 inflammasome and the subsequent release of IL-1β/IL-18, thereby alleviating the inflammatory response. Secondly, spermidine can reverse the intestinal flora imbalance induced by DSS, manifested as a decrease in the ratio of Firmicutes to Bacteroidetes, inhibition of Blautia enrichment, and restoration of the abundance of beneficial Muribaculaceae. These two pathways work together in synergy, jointly forming an integrated mechanism by which spermidine inhibits the NLRP3-mediated inflammatory response, promotes mitochondrial autophagy, and improves intestinal flora imbalance to alleviate DSS-induced colitis.

Article Snippet: The sections were then incubated at 4 °C overnight with primary antibodies targeting NLRP3 (1:500, Proteintech, 30109-1-AP, Chicago, IL, USA), PINK1 (1:500, Proteintech, 23274-1-AP, Chicago, IL, USA), and LC3 (1:500, Proteintech, 14600-1-AP, Chicago, IL, USA).

Techniques: Expressing, Activation Assay, Inhibition

Journal: Journal of Hematology & Oncology

Article Title: Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells

doi: 10.1186/s13045-015-0137-1

Figure Lengend Snippet: Oroxylin A inhibits the transcription of MDM2 via PTEN. (A) MCF-7 and HCT116 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was normalized to Renilla activity and expressed as luciferase/Renilla relative units. (B) H1299 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was measured. (C) Wt-PTEN plasmids were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into PC3M cells. Luciferase activity was measured. (D) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Luciferase activity was measured. (E) Cells were treated with oroxylin A for 48 h. Western blot assays were performed for PTEN. (F) Nucleus and cytosolic fractions were isolated after treatment and subjected to Western blot analysis for PTEN. (G) Immunofluorescence experiment performed in MCF-7 and HCT116 cells upon oroxylin A treatment using antibodies specific to PTEN and DAPI. (H) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Cells were then treated with 200 μΜ oroxylin A for 48 h. Luciferase activity was measured. (I) MCF-7 and HCT116 cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA, then incubated with 200 μM oroxylin A for 48 h. The mRNA expression of MDM2 was detected by Quantitative RT-PCR. (J) Cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA and incubated with 200 μM oroxylin A for 48 h. Western blot assays were performed for p53, MDM2, and PTEN. All the Western Blot bands were quantified. Bars, SD; * p < 0.05 or ** p < 0.01 versus non-treated control.

Article Snippet: The siRNAs targeting PTEN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Western Blot, Isolation, Immunofluorescence, Control, Incubation, Expressing, Quantitative RT-PCR

Journal: Journal of Hematology & Oncology

Article Title: Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells

doi: 10.1186/s13045-015-0137-1

Figure Lengend Snippet: Oroxylin A inhibited transcription of MDM2 by promoting SIRT3-regulated lipid phosphatase activity of PTEN. (A) Cells were treated with oroxylin A (OA) for 48 h. Nuclei were isolated and PTEN was immunoprecipitated using anti-FL SIRT3 antibody. Western blot assays were performed for PTEN and FL SIRT3. (B) Nuclei were isolated and acetylated PTEN was immunoprecipitated using anti-PTEN antibody. Western blot assays were performed for acetylated-lysine and PTEN. (C) Cells were co-transfected with MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and siRNA targeting SIRT3, then incubated with OA for 48 h. Luciferase activity was measured. (D, E) Cells were transfected with SIRT3 cDNA or treated with OA firstly. Then both were treated with NAM for 48 h. (D) Nuclei were isolated and Western blot assays were performed for MDM2, acetylated PTEN, and FL SIRT3. (E) Before treatments, MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) was co-transfected into cells. Luciferase activity was measured. (F) Cells were treated with sodium orthovanadate (SO) for 48 h. The mRNA expression of MDM2 was detected. (G) Cells were transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc or pGL3Basic-Mdm-T1-luc), and then treated with SO for 48 h. Luciferase activity was measured. (H) Cells were treated as that in (D). Lipid phosphatase activity of PTEN was assayed. (I, J, K) H1299 cells were treated with OA in/without the presence of tenovin-1for 48 h. (I) Nuclei were isolated and Western blot assays were performed for MDM2 and acetylated PTEN. (J) Before treatment, cells were transfected with MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc). Luciferase activity was measured. (K) Lipid phosphatase activity of PTEN was assayed. (L) Cells were transfected with siRNA targeting SIRT3 and incubated with OA for 48 h. Lipid phosphatase activity of PTEN was assayed. All the Western blot bands were quantified. Bars, SD; * p < 0.05 or ** p < 0.01 versus non-treated control.

Article Snippet: The siRNAs targeting PTEN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Isolation, Immunoprecipitation, Western Blot, Transfection, Luciferase, Plasmid Preparation, Incubation, Expressing, Control

Journal: Journal of Hematology & Oncology

Article Title: Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells

doi: 10.1186/s13045-015-0137-1

Figure Lengend Snippet: Oroxylin A-regulated glycolysis involved in the deacetylation of PTEN. (A) PC3M cells were co-transfected with wt-PTEN plasmids and siRNA SIRT3. Glucose uptake and lactate production were detected. (B) DU145 cells were co-transfected with PTEN siRNA and SIRT3 cDNA. Glucose uptake and lactate production were detected. (C, D) MCF-7 and HCT116 cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA, then incubated with 200 μM oroxylin A for 48 h. Lactate production (E) and glucose uptake (F) were detected. (E, F) MCF-7 and HCT116 cells were transfected with siRNA SIRT3, and then treated with 200 μM oroxylin A for 48 h, Lactate production (E) and glucose uptake (F) were detected. Bars, SD; * p < 0.05 or ** p < 0.01.

Article Snippet: The siRNAs targeting PTEN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Incubation

Journal: Journal of Hematology & Oncology

Article Title: Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells

doi: 10.1186/s13045-015-0137-1

Figure Lengend Snippet: Schematic diagram describing the effect of oroxylin A on the inhibition of p53 degradation through the suppression of PTEN-regulated MDM2 expression. Oroxylin A enhanced cellular SIRT3 level, causing the deacetylation of PTEN and promoting its lipid phosphatase activity. The activated PTEN suppressed the transcription of MDM2, which was responsible for the degradation of p53. As a result, oroxylin A inhibited p53 degradation and the p53-related glycolytic pathway.

Article Snippet: The siRNAs targeting PTEN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Expressing, Activity Assay

Journal: PLoS ONE

Article Title: The Role of PTEN in Chronic Growth Hormone-Induced Hepatic Insulin Resistance

doi: 10.1371/journal.pone.0068105

Figure Lengend Snippet: Mice were treated with daily hGH at 1 µg/g (controls received saline) for 3 weeks. (A) Fasting body weights were measured just before and after the completion of 3-week GH administration. (B) Plasma levels of Insulin were measured in mice without pre-sacrifice administration of insulin using ELISA Kit. (C) Hepatic Akt activity was examined in mice challenged by 10-minute insulin prior to sacrifice using Western blotting. (D) Hepatic Akt activity was examined in mice challenged by 10-min saline prior to sacrifice using Western blotting. (E) Hepatic PTEN expression was examined in mice challenged by 10-min insulin prior to sacrifice. (F) Hepatic PTEN expression was examined in mice challenged by 10-min saline prior to sacrifice. Liver lysates from mice challenged by 10-min insulin (G) or saline (H) prior to sacrifice were immunoprecipitated with antibody against the p85α subunit of PI3K and were blotted with antiserum specific, either for total tyrosine phosphorylation of p85, or for p85 phosphorylated at tyrosine 458 and tyrosine 508. The values in bar graphs were displayed as means ± standard errors.

Article Snippet: The small interfering RNAs targeting PTEN (siPTEN) were developed from Qiagen-Xeragon (Germantown, MD), the sense strand was 5′-GACUUGAAGGCGUAUACAGtt-3′, and the antisense strand was 5′-CUGUAUACGCCUUCAAGUCtt-3′.

Techniques: Saline, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Expressing, Immunoprecipitation, Phospho-proteomics

Journal: PLoS ONE

Article Title: The Role of PTEN in Chronic Growth Hormone-Induced Hepatic Insulin Resistance

doi: 10.1371/journal.pone.0068105

Figure Lengend Snippet: Mice were treated with bGH at 1 µg/g (controls received saline) daily for 3 weeks. (A) Fasting body weights were measured just before and after the completion of 3-week GH administration. (B) Hepatic Akt activity was examined in mice challenged by insulin 10 minutes prior to sacrifice using Western blotting. (C) Hepatic Akt activity was examined in mice challenged by 10-min saline prior to sacrifice. (D) Liver lysates from mice challenged by 10-min insulin prior to sacrifice were immunoprecipitated with antibody against PTEN, and were blotted with the same antibody. (E) Hepatic PTEN expression was examined in mice challenged by 10-min saline prior to sacrifice. Liver lysates from mice challenged by 10-min insulin (F) or saline (G) prior to sacrifice were immunoprecipitated with antibody against the p85α subunit of PI3K and were blotted with antiserum specific, either for total tyrosine phosphorylation of p85, or for p85 phosphorylated at tyrosine 458 and tyrosine 508. The values in bar graphs were displayed as means ± standard errors.

Article Snippet: The small interfering RNAs targeting PTEN (siPTEN) were developed from Qiagen-Xeragon (Germantown, MD), the sense strand was 5′-GACUUGAAGGCGUAUACAGtt-3′, and the antisense strand was 5′-CUGUAUACGCCUUCAAGUCtt-3′.

Techniques: Saline, Activity Assay, Western Blot, Immunoprecipitation, Expressing, Phospho-proteomics

Journal: PLoS ONE

Article Title: The Role of PTEN in Chronic Growth Hormone-Induced Hepatic Insulin Resistance

doi: 10.1371/journal.pone.0068105

Figure Lengend Snippet: (A) Fasting morning blood glucose of non-STZ and STZ group were measured by OneTouch® UltraVue™ glucometer prior to GH treatment. (B) Fasting body weights were measured before and after the completion of 3-week GH administration. (C) Hepatic Akt activity was examined in mice challenged by 10-minute insulin prior to sacrifice using Western blotting. (D) Hepatic PTEN expression was examined by Western blotting. (E) Liver lysates were immunoprecipitated with antibody against the p85α subunit of PI3K and were blotted with antiserum specific, either for total tyrosine phosphorylation of p85, or for p85 phosphorylated at tyrosine 458 and tyrosine 508. The values in bar graphs were displayed means ± standard errors.

Article Snippet: The small interfering RNAs targeting PTEN (siPTEN) were developed from Qiagen-Xeragon (Germantown, MD), the sense strand was 5′-GACUUGAAGGCGUAUACAGtt-3′, and the antisense strand was 5′-CUGUAUACGCCUUCAAGUCtt-3′.

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Phospho-proteomics

Journal: PLoS ONE

Article Title: The Role of PTEN in Chronic Growth Hormone-Induced Hepatic Insulin Resistance

doi: 10.1371/journal.pone.0068105

Figure Lengend Snippet: Mice were treated with daily hGH at 1 µg/g or saline for 3 weeks, and subjected to fasting in the last 3 days. (A) Fasting body weights were measured just before and after the completion of 3-week GH administration. (B) Plasma insulin levels were measured using ELISA Kit. (C) Hepatic Akt activity was examined using Western blotting. (D) Hepatic PTEN expression was examined using Western blotting. (E) Liver lysates were immunoprecipitated with antibody against the p85α subunit of PI3K and were blotted with antiserum specific, either for total tyrosine phosphorylation of p85, or for p85 phosphorylated at tyrosine 458 and tyrosine 508. The values in bar graphs were displayed means ± standard errors.

Article Snippet: The small interfering RNAs targeting PTEN (siPTEN) were developed from Qiagen-Xeragon (Germantown, MD), the sense strand was 5′-GACUUGAAGGCGUAUACAGtt-3′, and the antisense strand was 5′-CUGUAUACGCCUUCAAGUCtt-3′.

Techniques: Saline, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Expressing, Immunoprecipitation, Phospho-proteomics

Journal: PLoS ONE

Article Title: The Role of PTEN in Chronic Growth Hormone-Induced Hepatic Insulin Resistance

doi: 10.1371/journal.pone.0068105

Figure Lengend Snippet: (A) HepG2 cells were pretreated with GH or 30 min or 4 hours and harvested after insulin stimulation for 10 min. (B) Cells were transfected with wild type PTEN or PTEN mutant C124S, and harvested after insulin stimulation for 10 min. Densitometry absorbance values were averaged after they had been normalized to Akt for equal loading.

Article Snippet: The small interfering RNAs targeting PTEN (siPTEN) were developed from Qiagen-Xeragon (Germantown, MD), the sense strand was 5′-GACUUGAAGGCGUAUACAGtt-3′, and the antisense strand was 5′-CUGUAUACGCCUUCAAGUCtt-3′.

Techniques: Transfection, Mutagenesis

Journal: PLoS ONE

Article Title: The Role of PTEN in Chronic Growth Hormone-Induced Hepatic Insulin Resistance

doi: 10.1371/journal.pone.0068105

Figure Lengend Snippet: Cells were transfected with siRNA targeted for PTEN (A), PTEN mutant C124S (B) or wtPTEN (C), and were stimulated with GH and Insulin as stated in Methods and materials. Densitometry absorbance values were averaged after they had been normalized to Akt for equal loading.

Article Snippet: The small interfering RNAs targeting PTEN (siPTEN) were developed from Qiagen-Xeragon (Germantown, MD), the sense strand was 5′-GACUUGAAGGCGUAUACAGtt-3′, and the antisense strand was 5′-CUGUAUACGCCUUCAAGUCtt-3′.

Techniques: Transfection, Mutagenesis