Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: MiR-106a-5p induces ferroptosis by targeting STAT3 in breast cancer cells. ( A ) The interaction of miR-106a-5p and STAT3 3’ UTR was identified by bioinformatic analysis using Targetscan ( http://www.targetscan.org/vert_72/ ). ( B – D ) The MDA-MB-231 and T47D cells were treated with the miR-106a-5p mimic or control mimic. ( B ) The luciferase activities of wild type STAT3 (STAT3 WT) and STAT3 with the miR-106a-5p-binding site mutant (STAT3 MUT) were determined by luciferase reporter gene assays in the cell. ( C ) The mRNA expression of STAT3 was analyzed by qPCR in the cells. ( D ) The protein expression of STAT3 and β-actin was tested by Western blot analysis in the cells. ( E ) The MDA-MB-231 and T47D cells were treated control shRNA, circRHOT1 shRNA, or co-treated with circRHOT1 shRNA and miR-106a-5p inhibitor. The protein expression of STAT3 and β-actin was assessed by Western blot analysis in the cells. ( E , F ) The MDA-MB-231 and T47D cells were treated with 5 mmol/L erastin, co-treated with 5 mmol/L erastin and miR-106a-5p mimic, or o-treated with 5 mmol/L erastin, miR-106a-5p mimic, and pcDNA.1-STAT3. The cell growth was analyzed by MTT assays. ( G – I ) The MDA-MB-231 and T47D cells were treated control shRNA, miR-106a-5p mimic, or co-treated with miR-106a-5p mimic and pcDNA.1-STAT3. ( G ) The levels of iron were analyzed by Iron Assay Kit. ( H ) The levels of ROS were measure by flow cytometry analysis in the cells. ( I ) The expression of GPX4, SLC7A11, and β-actin was measured by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: Luciferase, Binding Assay, Mutagenesis, Expressing, Western Blot, shRNA, Iron Assay, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: CircRHOT1 contributes to breast cancer progression by miR-106a-5p/STAT3 axis. ( A – D ) The MDA-MB-231 and T47D cells were treated control shRNA, circRHOT1 shRNA, or co-treated with circRHOT1 shRNA and miR-106a-5p inhibitor or pcDNA.1-STAT3. ( A , B ) The cell viability was measured by MTT assays in the cells. ( C , D ) The cell apoptosis was measure by flow cytometry analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: shRNA, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: CircRHOT1 promotes the tumor growth of breast cancer in vivo . ( A – E ) The effect of circRHOT1 on tumor growth of breast cancer cells in vivo was analyzed by nude mice tumorigenicity assay by injected with the MDA-MB-231 cells treated with control shRNA or circRHOT1 shRNA. ( A ) Representative images of dissected tumors from nude mice were presented. ( B ) The average tumor volume was calculated and shown. ( C ) The average tumor weight was calculated and shown. ( D ) The expression levels of miR-106a-5p were measured by qPCR in the tumor tissues of the mice. ( E ) The protein expression of STAT3 and β-actin was assessed by Western blot analysis in the tumor tissues of the mice. N = 5. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: In Vivo, Tumorigenicity Assay, Injection, shRNA, Expressing, Western Blot

Journal: World Journal of Gastrointestinal Oncology

Article Title: Propofol induces ferroptosis and inhibits malignant phenotypes of gastric cancer cells by regulating miR-125b-5p/STAT3 axis

doi: 10.4251/wjgo.v13.i12.2114

Figure Lengend Snippet: Propofol represses signal transducer and activator of transcription (STAT)3 expression by upregulating miR-125b-5p in gastric cancer cells. A: SGC7901 and BGC823 cells were treated with propofol (10 µmol/L). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measured expression of miR-125b-5p. B: The binding site of miR-125b-5p and STAT3 3’ UTR was identified by bioinformatic analysis using Targetscan ( http://www.targetscan.org/vert_72/ ). C–E: SGC7901 and BGC823 cells were treated with the miR-125b-5p mimic or control mimic. C and D: Luciferase reporter gene assays determined the luciferase activities. E: qRT-PCR analyzed mRNA expression of STAT3. F: SGC7901 and BGC823 cells were treated with propofol, or cotreated with propofol and miR-125b-5p inhibitor. Western blotting assessed protein expression of STAT3 and β-actin. n = 3, mean ± SD, b P < 0.01.

Article Snippet: The pcDNA3.1-STAT3 overexpression vector, miR-125b-5p mimic, miR-125b-5p inhibitor, and corresponding control were purchased from Genscript Biotech Corporation (Nanjing, China) and GenePharma Co. Ltd. (Shanghai, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Luciferase, Western Blot

Journal: World Journal of Gastrointestinal Oncology

Article Title: Propofol induces ferroptosis and inhibits malignant phenotypes of gastric cancer cells by regulating miR-125b-5p/STAT3 axis

doi: 10.4251/wjgo.v13.i12.2114

Figure Lengend Snippet: Propofol enhances ferroptosis by targeting signal transducer and activator of transcription (STAT)3 in gastric cancer cells. A: SGC7901 and BGC823 cells were treated with 5 mmol/L erastin, cotreated with 5 mmol/L erastin and propofol, or cotreated with 5 mmol/L erastin, propofol, and pcDNA.1-STAT3. MTT assays measured cell growth. B–D: SGC7901 and BGC823 cells were treated with propofol, or cotreated with propofol and pcDNA.1-STAT3. B: Iron Assay Kit analyzed the levels of iron; C: Flow cytometry analysis tested the levels of ROS; and D: Iron Assay Kit analyzed the levels of Fe 2+ . n = 3, mean ± SD, b P < 0.01, c P < 0.001.

Article Snippet: The pcDNA3.1-STAT3 overexpression vector, miR-125b-5p mimic, miR-125b-5p inhibitor, and corresponding control were purchased from Genscript Biotech Corporation (Nanjing, China) and GenePharma Co. Ltd. (Shanghai, China).

Techniques: Iron Assay, Flow Cytometry

Journal: World Journal of Gastrointestinal Oncology

Article Title: Propofol induces ferroptosis and inhibits malignant phenotypes of gastric cancer cells by regulating miR-125b-5p/STAT3 axis

doi: 10.4251/wjgo.v13.i12.2114

Figure Lengend Snippet: Propofol attenuates gastric cancer progression by miR-125b-5p/STAT3 axis. A–C: SGC7901 and BGC823 cells were treated propofol, or cotreated with propofol and miR-125b-5p inhibitor or pcDNA.1-STAT3. A and B: MTT assays analyzed the cell viability; C: Flow cytometry measured apoptosis. n = 3, mean ± SD, b P < 0.01.

Article Snippet: The pcDNA3.1-STAT3 overexpression vector, miR-125b-5p mimic, miR-125b-5p inhibitor, and corresponding control were purchased from Genscript Biotech Corporation (Nanjing, China) and GenePharma Co. Ltd. (Shanghai, China).

Techniques: Flow Cytometry

Journal: World Journal of Gastrointestinal Oncology

Article Title: Propofol induces ferroptosis and inhibits malignant phenotypes of gastric cancer cells by regulating miR-125b-5p/STAT3 axis

doi: 10.4251/wjgo.v13.i12.2114

Figure Lengend Snippet: Propofol attenuates growth of gastric cancer cells in vivo . The nude mice were injected with SGC7901 cells and intraperitoneally treated with propofol (50 mg/kg). A: Tumor tissues; B: Tumor volume; and C: Tumor weight; D: Expression of miR-125b-5p was analyzed by quantitative reverse transcription polymerase chain reaction. E: Protein expression of STAT3 was detected by western blotting. F: Protein expression of GPX4 and SLC7A11 was measured by western blotting. n = 5, mean ± SD, b P < 0.01.

Article Snippet: The pcDNA3.1-STAT3 overexpression vector, miR-125b-5p mimic, miR-125b-5p inhibitor, and corresponding control were purchased from Genscript Biotech Corporation (Nanjing, China) and GenePharma Co. Ltd. (Shanghai, China).

Techniques: In Vivo, Injection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-140-5p regulates the proliferation, apoptosis and inflammation of RA FLSs by repressing STAT3

doi: 10.3892/etm.2020.9602

Figure Lengend Snippet: STAT3 is a direct target of miR-140. (A) Graphical representation of the conserved miR-140 binding motif at the STAT3 3'-UTR. The complementary sequences to the seed regions of miR-140 and corresponding sequence of the 3'-UTR of STAT3. (B) The luciferase activity exhibited by the reporter constructs, containing either the WT or MUT human STAT3 3'-UTR after miR-140 transfection. The observed luciferase activity was normalized to that of β-galactosidase. Overexpression of miR-140 markedly decreased the relative luciferase activity in the WT 3'-UTR, but not the MUT 3'-UTR, of STAT3 mRNA. All groups were normalized to the WT + control group (100%). (C) Western blotting and (D) RT-qPCR analysis were used to evaluate the STAT3 protein and mRNA expression levels after transfection of RA FLSs with miR-140 or miR-negative control. All groups were normalized to the control group (100%). (E) Increased expression levels of STAT3 in RA FLSs was observed by RT-qPCR, compared to that of healthy FLSs. All groups were normalized to the normal FLS group (100%). Data represents the mean ± SD. * P<0.05, ** P<0.01 vs. the corresponding control. FLS, fibroblast-like synoviocyte; miR, microRNA; MUT, mutant; RA, rheumatoid arthritis; RT-qPCR, reverse transcription-quantitative PCR; UTR, untranslated region; WT, wild-type.

Article Snippet: The product was inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate STAT3 expression vectors pcDNA3.1-SOX11.

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Transfection, Over Expression, Western Blot, Quantitative RT-PCR, Expressing, Negative Control, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-140-5p regulates the proliferation, apoptosis and inflammation of RA FLSs by repressing STAT3

doi: 10.3892/etm.2020.9602

Figure Lengend Snippet: STAT3 was overexpressed in cells. Cells were transfected with or without STAT3-expressing vector or empty vector. Overexpression of STAT3 in the cells was then detected using western blotting at 48 h post-transfection. NC, negative control.

Article Snippet: The product was inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate STAT3 expression vectors pcDNA3.1-SOX11.

Techniques: Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-140-5p regulates the proliferation, apoptosis and inflammation of RA FLSs by repressing STAT3

doi: 10.3892/etm.2020.9602

Figure Lengend Snippet: STAT3 restored the regulatory role of miR-140 in RA FLSs. STAT3 expression levels were detected in cells using (A) western blotting and (B) RT-qPCR following miR-140 and STAT3 transfections. All groups were normalized to the control group (100%). The proliferative ability of cells expressing STAT3 and miR-140, miR-140 alone, or control was measured at 48 h post-transfection using the (C) BrdU incorporation assays and (D) MTT assays. (E) The apoptotic rate of RA FLSs in each group was determined by annexin V-FITC/PI flow cytometry. Early apoptotic cells and later apoptotic cells are indicated in the top right and bottom right quadrants, respectively, in each plot. Quantitative analysis of the apoptotic rate of RA FLSs in two groups is displayed in the right panel. (F) Western blotting was carried out to assess the expression levels of pro-inflammatory cytokines regulated by the miR-140 and STAT3 plasmid transfections. (G) RT-qPCR was performed to examine the gene expression of cytokines from the RA FLSs after transfection. All groups were normalized to the control group (100%). Data represents the mean ± SD. * P<0.05, ** P<0.01 vs. the control group; # P<0.05, ## P<0.01 vs. the miR-140 group. FLS, fibroblast-like synoviocyte; IL, interleukin; miR, microRNA; PI, propidium iodide; RA, rheumatoid arthritis; RT-qPCR, reverse transcription-quantitative PCR; TNF, tumor necrosis factor.

Article Snippet: The product was inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate STAT3 expression vectors pcDNA3.1-SOX11.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, BrdU Incorporation Assay, Cytometry, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: A role for annexin A2 in scaffolding the peroxiredoxin 2–STAT3 redox relay complex

doi: 10.1038/s41467-020-18324-9

Figure Lengend Snippet: a Bimolecular fluorescence complementation: STAT3-LN interacts more strongly with Prx2-LC than with Prx1-LC (bars 2-3). Fluorescence complementation between STAT3-LN and Prx2-LC can be competed with Prx2-SBP (bars 4–6), but not with Prx1-SBP (bars 7–9). The negative control (bar 1) indicates non-specific complementation between unfused C- and N-terminal domains of mLumin (LC and LN, respectively). The immunoblots visualize the stepwise increase of Prx2-SBP and Prx1-SBP expression. Based on n = 6 independent experiments with n = 6 technical replicates each. IB: immunoblot. Source data are provided as a Source data file. b Co-precipitation: SBP-STAT3 co-precipitates Prx2 (left panels), and Prx2-SBP co-precipitates STAT3 (right panels). Blots are representative of n = 5 independent experiments. PD pulldown; IB immunoblot. Source data are provided as a Source data file. c Proximity ligation: Endogenous (untagged) Prx2 and STAT3 form a complex. Representative images (left panels) show PLA foci in red and DAPI in blue. The Prx2-Prx2 interaction (homo-oligomer formation) was used as a positive control, and single antibody experiments were included as negative controls. Scale bar: 10 µm. The bar graph (right panel) quantifies the number of PLA foci per cell ( n = 50–60 cells in two technical replicates). The results are representative of n = 3 independent experiments. All bar charts in this figure represent the mean ± SD. * p < 0.05; *** p < 0.001; **** p < 0.0001; ns not significant; based on a two-tailed unpaired t -test.

Article Snippet: Expression plasmids used in this study were pcDNA3.1(-) STAT3-HA-LN151, pLPCX STAT3-HA-LN151, pcDNA3.1(-) Prx2-MYC-LC151, pLPCX Prx2-MYC-LC151, pcDNA3.1(-) Prx1-MYC-LC151, pcDNA3.1(-) MYC-LC151, pcDNA3.1(-) HA-LN151, pcDNA3.1(-) NTD-HA-LN151, pcDNA3.1(-) Cerulean-STAT3, pcDNA3.1(-) Prx2-Venus, pcDNA3.1(-) Venus-Prx2, pcDNA3.1(-) Cerulean-5-Venus, pcDNA3.1(-) Cerulean-P2A-Venus, pcDNA3.1(-) Prx2-SBP, pcDNA3.1(-) SBP-STAT3, pcDNA3.1(-) SBP-NTD and pcDNA3.1(-) Annexin A2, and pGL4.47(luc2P/SIE/Hygro) (Promega).

Techniques: Fluorescence, Negative Control, Western Blot, Expressing, Ligation, Positive Control, Two Tailed Test

Journal: Nature Communications

Article Title: A role for annexin A2 in scaffolding the peroxiredoxin 2–STAT3 redox relay complex

doi: 10.1038/s41467-020-18324-9

Figure Lengend Snippet: a The STAT3-Prx2 interaction can be competed with the NTD of STAT3. HEK293 MSR cells stably expressing Prx2-LC were co-transfected with STAT3-LN and increasing concentrations of SBP-tagged NTD. The bar charts in this figure show the mean ± SD from n = 6 independent experiments with n = 6 technical replicates each. b The NTD of STAT3 co-precipitates endogenous Prx2. The immunoblot is representative of n = 3 independent experiments. PD pulldown; IB immunoblot. Source data are provided as a Source data file. c Point mutations at the NTD surface disrupt the interaction between the NTD and Prx2. HEK293 MSR cells stably expressing Prx2-LC were transfected with NTD-LN, NTD(L78R)-LN or NTD(W37A)-LN. The corresponding immunoblot is shown in Supplementary Fig. 2a. The bar charts in this figure show the mean ± SD from n = 4 independent experiments with n = 6 technical replicates each. d Point mutations at the NTD surface disrupt the interaction between full-length STAT3 and Prx2. HEK293 MSR cells stably expressing Prx2-LC were transfected with STAT3-LN, STAT3(L78R)-LN or STAT3(W37A)-LN. The corresponding immunoblot is shown in Supplementary Fig. 2b. The bar charts in this figure show the mean ± SD from n = 4 independent experiments with n = 6 technical replicates each. e Point mutations at the NTD surface disrupt the formation of STAT3 oxidation products (top panel) and of Prx2-STAT3 disulfide exchange intermediates (second panel from top). The blots are representative of n = 3 independent experiments. PD pulldown; IB immunoblot. Source data are provided as a Source Data file. * P < 0.05; *** P < 0.001; **** P < 0.0001; ns not significant; based on a two-tailed unpaired t -test.

Article Snippet: Expression plasmids used in this study were pcDNA3.1(-) STAT3-HA-LN151, pLPCX STAT3-HA-LN151, pcDNA3.1(-) Prx2-MYC-LC151, pLPCX Prx2-MYC-LC151, pcDNA3.1(-) Prx1-MYC-LC151, pcDNA3.1(-) MYC-LC151, pcDNA3.1(-) HA-LN151, pcDNA3.1(-) NTD-HA-LN151, pcDNA3.1(-) Cerulean-STAT3, pcDNA3.1(-) Prx2-Venus, pcDNA3.1(-) Venus-Prx2, pcDNA3.1(-) Cerulean-5-Venus, pcDNA3.1(-) Cerulean-P2A-Venus, pcDNA3.1(-) Prx2-SBP, pcDNA3.1(-) SBP-STAT3, pcDNA3.1(-) SBP-NTD and pcDNA3.1(-) Annexin A2, and pGL4.47(luc2P/SIE/Hygro) (Promega).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: A role for annexin A2 in scaffolding the peroxiredoxin 2–STAT3 redox relay complex

doi: 10.1038/s41467-020-18324-9

Figure Lengend Snippet: a Co-precipitation: Both Prx2 (upper panels) and STAT3 (lower panels) co-precipitate AnxA2. The blots are representative of n = 3 independent experiments. PD pulldown; IB immunoblot. Source data are provided as a Source data file. b Proximity ligation: AnxA2 interacts with Prx2 and STAT3 (upper panels). S100A10 interacts with AnxA2 (positive control), but not with Prx2 or STAT3 (middle panels). Single antibody experiments are included as negative controls. Representative images show PLA foci in red and DAPI in blue. Scale bars: 10 µm. The bar graphs (lower panels) quantify the number of PLA foci per cell ( n = 50–60 cells in n = 2 technical replicates). The results are representative of n = 3 independent experiments. c Depletion of AnxA2 by siRNA diminishes the association between Prx2 and STAT3. Co-precipitation of Prx2 by SBP-STAT3 (left panels) and co-precipitation of STAT3 by Prx2-SBP (right panels). The blots are representative of n = 3 independent experiments. PD pulldown; IB immunoblot. Source data are provided as a Source data file. d Deletion of AnxA2 compromises the Prx2-STAT3 interaction as detected by PLA. Representative images show PLA foci in red and DAPI in blue (left panels). Scale bar: 10 µm. The bar graphs (right panels) show the frequency of PLA foci in AnxA2 KO cells relative to wild type cells ( n = 40–50 cells in n = 3 technical replicates), using two independent clones of AnxA2 KO cells. The interaction of AnxA2 with Prx2 or STAT3 in AnxA2 KO cells represents the background signal. The results are representative of n = 3 independent experiments. All bar charts in this figure represent the mean ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns not significant; based on a two-tailed unpaired t -test.

Article Snippet: Expression plasmids used in this study were pcDNA3.1(-) STAT3-HA-LN151, pLPCX STAT3-HA-LN151, pcDNA3.1(-) Prx2-MYC-LC151, pLPCX Prx2-MYC-LC151, pcDNA3.1(-) Prx1-MYC-LC151, pcDNA3.1(-) MYC-LC151, pcDNA3.1(-) HA-LN151, pcDNA3.1(-) NTD-HA-LN151, pcDNA3.1(-) Cerulean-STAT3, pcDNA3.1(-) Prx2-Venus, pcDNA3.1(-) Venus-Prx2, pcDNA3.1(-) Cerulean-5-Venus, pcDNA3.1(-) Cerulean-P2A-Venus, pcDNA3.1(-) Prx2-SBP, pcDNA3.1(-) SBP-STAT3, pcDNA3.1(-) SBP-NTD and pcDNA3.1(-) Annexin A2, and pGL4.47(luc2P/SIE/Hygro) (Promega).

Techniques: Western Blot, Ligation, Positive Control, Clone Assay, Two Tailed Test

Journal: Nature Communications

Article Title: A role for annexin A2 in scaffolding the peroxiredoxin 2–STAT3 redox relay complex

doi: 10.1038/s41467-020-18324-9

Figure Lengend Snippet: a H 2 O 2 -induced STAT3 oxidation is diminished upon depletion of AnxA2 in HEK293 MSR cells. The formation of covalent (disulfide-linked) STAT3 dimers and tetramers in response to increasing concentrations of exogenously applied H 2 O 2 is visualized by immunoblotting on non-reducing SDS-PAGE. b The same experiment as in a , performed in U2OS cells. c The formation of Prx2-STAT3 disulfide exchange intermediates, representing the transfer of oxidizing equivalents from Prx2 to STAT3, is largely abolished upon depletion of AnxA2 in HEK293 MSR cells. d H 2 O 2 -induced STAT3 oxidation is largely abolished upon deletion of AnxA2 (AnxA2 KO) in HEK293 MSR cells. e Re-expression of ectopic AnxA2 in AnxA2 KO cells recovers H 2 O 2 -induced STAT3 oxidation. All immunoblots shown in this figure are representative of n = 3 independent experiments. IB immunoblot; NR/R Non-reducing/Reducing gel electrophoresis. All source data for this figure are provided as a Source data file.

Article Snippet: Expression plasmids used in this study were pcDNA3.1(-) STAT3-HA-LN151, pLPCX STAT3-HA-LN151, pcDNA3.1(-) Prx2-MYC-LC151, pLPCX Prx2-MYC-LC151, pcDNA3.1(-) Prx1-MYC-LC151, pcDNA3.1(-) MYC-LC151, pcDNA3.1(-) HA-LN151, pcDNA3.1(-) NTD-HA-LN151, pcDNA3.1(-) Cerulean-STAT3, pcDNA3.1(-) Prx2-Venus, pcDNA3.1(-) Venus-Prx2, pcDNA3.1(-) Cerulean-5-Venus, pcDNA3.1(-) Cerulean-P2A-Venus, pcDNA3.1(-) Prx2-SBP, pcDNA3.1(-) SBP-STAT3, pcDNA3.1(-) SBP-NTD and pcDNA3.1(-) Annexin A2, and pGL4.47(luc2P/SIE/Hygro) (Promega).

Techniques: Western Blot, SDS Page, Expressing, Nucleic Acid Electrophoresis

Journal: Nature Communications

Article Title: A role for annexin A2 in scaffolding the peroxiredoxin 2–STAT3 redox relay complex

doi: 10.1038/s41467-020-18324-9

Figure Lengend Snippet: a STAT3 oxidation is associated with membranes. HEK293 MSR cells were pulsed with 100 µM H 2 O 2 for 2 min and then fractionated into cytosol, membranes, and nuclei. The corresponding reducing immunoblot is shown in Supplementary Fig. . The blots are representative of n = 3 independent experiments. IB immunoblot. Source data are provided as a Source data file. b Membrane-associated STAT3 oxidation depends on AnxA2. HEK293 MSR wild type and AnxA2 KO cells were fractionated into cytosol, membranes and nuclei. The corresponding reducing immunoblot is shown in Supplementary Fig. . The blots are representative of n = 3 independent experiments. IB immunoblot. Source data are provided as a Source data file. c AnxA2 deficiency increases STAT3-dependent transcription from the serum inducible element (SIE) promotor. STAT3 signaling was induced by either IL-6 or OSM treatment and SIE promotor activity was measured with a luciferase reporter. Bars represent the mean (±SD) of n = 3 biological replicates. ** p < 0.01; *** p < 0.001; based on a two-tailed unpaired t -test.

Article Snippet: Expression plasmids used in this study were pcDNA3.1(-) STAT3-HA-LN151, pLPCX STAT3-HA-LN151, pcDNA3.1(-) Prx2-MYC-LC151, pLPCX Prx2-MYC-LC151, pcDNA3.1(-) Prx1-MYC-LC151, pcDNA3.1(-) MYC-LC151, pcDNA3.1(-) HA-LN151, pcDNA3.1(-) NTD-HA-LN151, pcDNA3.1(-) Cerulean-STAT3, pcDNA3.1(-) Prx2-Venus, pcDNA3.1(-) Venus-Prx2, pcDNA3.1(-) Cerulean-5-Venus, pcDNA3.1(-) Cerulean-P2A-Venus, pcDNA3.1(-) Prx2-SBP, pcDNA3.1(-) SBP-STAT3, pcDNA3.1(-) SBP-NTD and pcDNA3.1(-) Annexin A2, and pGL4.47(luc2P/SIE/Hygro) (Promega).

Techniques: Western Blot, Activity Assay, Luciferase, Two Tailed Test

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SNHG20 promotes colorectal cancer cell proliferation, migration and invasion via miR-495/STAT3 axis

doi: 10.3892/mmr.2020.11669

Figure Lengend Snippet: Primers used for reverse transcription-quantitative PCR analysis.

Article Snippet: Lentiviral knockdown vector specifically targeting SNHG20 [small interfering (si) RNA-SNHG20] and negative control (si-NC) were provided by Shanghai GenePharma Co., Ltd. miR-495 mimics, miR-495 inhibitor and their NCs were purchased from Shanghai GenePharma Co., Ltd. STAT3 overexpression plasmid [pcDNA3.1(+) STAT3] and its NC [pcDNA3.1(+)] were constructed by Shanghai GenePharma Co., Ltd. Before transfection, cells were subcultured in 6-well plates (Corning, Inc.) ~50% confluence (3×10 5 ).

Techniques:

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SNHG20 promotes colorectal cancer cell proliferation, migration and invasion via miR-495/STAT3 axis

doi: 10.3892/mmr.2020.11669

Figure Lengend Snippet: STAT3 expression is negatively regulated by miR-495. (A) Bioinformatics analysis of predicted binding sites between miR-495 and STAT3. (B) The relative expression levels of STAT3 were significantly increased in CRC tissues. (C) Pearson's correlation analysis showed an inverse correlation between miR-495 and STAT3 expression in CRC tissues. (D) Luciferase reporter assays indicated that transfection with the miR-495 mimic significantly reduced the luciferase activity of STAT3-WT in HCT116 and SW480 cells. Overexpression of miR-495 significantly reduced the expression of STAT3 at the (E) mRNA and (F) protein levels in HCT116 and SW480 cells. ***P<0.001. CRC, colorectal cancer; miR, microRNA; si-, small interfering RNA; NC, negative control; WT, wild-type; MUT, mutant.

Article Snippet: Lentiviral knockdown vector specifically targeting SNHG20 [small interfering (si) RNA-SNHG20] and negative control (si-NC) were provided by Shanghai GenePharma Co., Ltd. miR-495 mimics, miR-495 inhibitor and their NCs were purchased from Shanghai GenePharma Co., Ltd. STAT3 overexpression plasmid [pcDNA3.1(+) STAT3] and its NC [pcDNA3.1(+)] were constructed by Shanghai GenePharma Co., Ltd. Before transfection, cells were subcultured in 6-well plates (Corning, Inc.) ~50% confluence (3×10 5 ).

Techniques: Expressing, Binding Assay, Luciferase, Transfection, Activity Assay, Over Expression, Small Interfering RNA, Negative Control, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SNHG20 promotes colorectal cancer cell proliferation, migration and invasion via miR-495/STAT3 axis

doi: 10.3892/mmr.2020.11669

Figure Lengend Snippet: SNHG20 regulates miR-495/STAT3 signaling in CRC. (A-C) SNHG20 knockdown decreased the mRNA and protein expression levels of STAT3, which was reversed by the addition of the miR-495 inhibitor in HCT116 and SW480 cells. (D) Pearson's correlation analysis indicated that SNHG20 expression was positively correlated with STAT3 expression in CRC tissues. (E) Reverse transcription-quantitative PCR and (F) western blotting indicated that the mRNA and protein levels of STAT3 were significantly increased following transfection of the cells with pcDNA3.1(+) STAT3. SNHG20 knockdown decreased the (G) mRNA and (H) protein expression levels of STAT3, which could be reversed by STAT3 overexpression. ***P<0.001. SNHG20, small nucleolar RNA host gene 20; CRC, colorectal cancer; miR, microRNA; si-, small interfering RNA; NC, negative control.

Article Snippet: Lentiviral knockdown vector specifically targeting SNHG20 [small interfering (si) RNA-SNHG20] and negative control (si-NC) were provided by Shanghai GenePharma Co., Ltd. miR-495 mimics, miR-495 inhibitor and their NCs were purchased from Shanghai GenePharma Co., Ltd. STAT3 overexpression plasmid [pcDNA3.1(+) STAT3] and its NC [pcDNA3.1(+)] were constructed by Shanghai GenePharma Co., Ltd. Before transfection, cells were subcultured in 6-well plates (Corning, Inc.) ~50% confluence (3×10 5 ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SNHG20 promotes colorectal cancer cell proliferation, migration and invasion via miR-495/STAT3 axis

doi: 10.3892/mmr.2020.11669

Figure Lengend Snippet: SNHG20/miR-495/STAT3 axis regulates CRC progression. (A) MTS and (B) colony formation assays suggested that SNHG20 knockdown decreased CRC cell viability, which could be reversed by STAT3 overexpression. (C) Transwell assays indicated that SNHG20 knockdown inhibited migration and invasion of CRC cells, which could be reversed by STAT3 overexpression. *P<0.05 and ***P<0.001. SNHG20, Small nucleolar RNA host gene 20; CRC, colorectal cancer; miR, microRNA; si-, small interfering RNA; NC, negative control.

Article Snippet: Lentiviral knockdown vector specifically targeting SNHG20 [small interfering (si) RNA-SNHG20] and negative control (si-NC) were provided by Shanghai GenePharma Co., Ltd. miR-495 mimics, miR-495 inhibitor and their NCs were purchased from Shanghai GenePharma Co., Ltd. STAT3 overexpression plasmid [pcDNA3.1(+) STAT3] and its NC [pcDNA3.1(+)] were constructed by Shanghai GenePharma Co., Ltd. Before transfection, cells were subcultured in 6-well plates (Corning, Inc.) ~50% confluence (3×10 5 ).

Techniques: Over Expression, Migration, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: Long non-coding RNA SNHG20 promotes colorectal cancer cell proliferation, migration and invasion via miR-495/STAT3 axis

doi: 10.3892/mmr.2020.11669

Figure Lengend Snippet: SNHG20 knockdown inhibits CRC growth and metastasis in vivo . (A) The mean volume of the xenograft tumor in the SNHG20-knockdown group was significantly lower than that of the NC group. (B) The average weight of the xenograft tumors in the SNHG20-knockdown group was significantly lower than that noted in the NC group. (C) The longest tumor diameter of the tumors in the SNHG20-knockdown group was significantly smaller than that in the NC group. (D) The relative expression levels of miR-495 in the SNHG20-knockdown group were significantly increased compared with those in the NC group. (E) The relative expression levels of STAT3 in the SNHG20-knockdown group were significantly reduced compared with those in the NC group. (F) Bioluminescence imaging analyses indicated that SNHG20 knockdown significantly inhibited CRC cell lung metastatic ability compared with that of the NC group. (G) SNHG20 knockdown resulted in a significant reduction of the number of metastatic pulmonary nodules compared with that of the NC group. *P<0.05, **P<0.01 and ***P<0.001 vs. si-SNHG20 or as indicated. SNHG20, small nucleolar RNA host gene 20; CRC, colorectal cancer; miR, microRNA; si-, small interfering RNA; NC, negative control.

Article Snippet: Lentiviral knockdown vector specifically targeting SNHG20 [small interfering (si) RNA-SNHG20] and negative control (si-NC) were provided by Shanghai GenePharma Co., Ltd. miR-495 mimics, miR-495 inhibitor and their NCs were purchased from Shanghai GenePharma Co., Ltd. STAT3 overexpression plasmid [pcDNA3.1(+) STAT3] and its NC [pcDNA3.1(+)] were constructed by Shanghai GenePharma Co., Ltd. Before transfection, cells were subcultured in 6-well plates (Corning, Inc.) ~50% confluence (3×10 5 ).

Techniques: In Vivo, Expressing, Imaging, Small Interfering RNA, Negative Control