Journal: Nature cell biology

Article Title: Phosphorylation of STIM1 Underlies Suppression of Store-operated Calcium Entry During Mitosis

doi: 10.1038/ncb1995

Figure Lengend Snippet: a) SOCE was measured in HeLa cells by treating cells with thapsigargin (Tg; 2 μM) in the continuous presence of 1.8 mM extracellular Ca2+ as indicated (except green trace). For controls, interphase cells were selected from an asynchronous population based on their flat, well-spread morphology (black trace). For comparison, cells treated with thapsigargin in the presence of nominally Ca2+-free external solution failed to exhibit a sustained SOCE response (green trace). Nocodazole (NZL)-arrested mitotic cells were selected based on their spherical morphology (red trace), and flat interphase cells were also selected from the same population of nocodazole-treated cells (blue trace). b) SOCE was measured in HEK293 cells by treating cells with thapsigargin in nominally Ca2+-free external solution, followed by restoration of 1.8 mM external Ca2+ as indicated. Interphase cells (black trace) were selected from asynchronous populations, whereas mitotic cells were arrested by nocodazole treatment (red trace) or were identified from asynchronous populations based on their round morphology (blue trace). Each trace represents the averaged response from 20–30 cells from a single experiment. c) The peak increase in fluorescence ratio above baseline following Ca2+ add-back was calculated and averaged for each cell from experiments as described in panel b. Interphase: n = 90 cells, 3 coverslips; Mitotic (nocodazole-arrested): n = 88 cells, 3 coverslips. ‘*’ indicates statistically significant difference (t-test; p < 0.0001). Error bars represent S.E.M. d) Total protein lysates from asynchronous or nocodazole-arrested mitotic HeLa (lanes 1 and 2) and HEK293 (lanes 3 and 4) cells were analyzed by Western blot with anti-STIM1 (upper panel), anti-Orai1 (middle panel) and anti-β-actin (lower panel) antibodies. The same blot was stripped and reprobed between each antibody. Also analyzed were lysates from asynchronous HEK293 cells treated with control siRNA or Orai1-specific siRNA (Lanes 5 and 6). Full scans of these blots are shown in Supplemental Figure 6a. Note the upward shift in apparent molecular weight of STIM1 from mitotic compared to asynchronous samples. Orai1 appeared as a series of differentially glycosylated bands between 36 and 50 kDa. The multiple banding pattern of Orai1 is likely due to the presence of multiple patterns of N-glycosylation, because treatment of cell lysates with N-glycosidase F causes a loss of this banding pattern and the appearance of distinct Orai1 bands (Supplemental Figure 1). e) SOCE responses in HEK293 cells upon 1.8 mM Ca2+ add-back following store depletion with thapsigargin (Tg; 2 μM). Black trace: interphase cells selected from an asynchronous population; blue trace: interphase cells selected from an asynchronous, Orai1 siRNA-treated population; red trace: nocodazole-arrested mitotic cells. Each trace represents the averaged response of 20–30 cells from a single experiment. f) Western blot analysis of Orai1 protein expression in total lysates from wildtype (WT; lanes 1 and 2) or eYFP-STIM1 and Orai1 overexpressing (S1+O1; lanes 3 and 4) asynchronous and mitotic HEK293 cells. Because of the substantial difference in protein expression levels in wildtype and overexpressing samples, a long and short exposure of the same blot are shown. β-actin is shown as a loading control. A full scan of the Orai1 blot is shown in Supplemental Figure 6b. g) SOCE responses in interphase (black trace) or mitotic (red trace) eYFP-STIM1 and Orai1 overexpressing HEK293 cells as described in panel e, except Ca2+ add-back was 1.0 mM. h) The peak increase in fluorescence ratio above baseline following Ca2+ add-back was calculated and averaged for each cell from experiments as described in panel c. Interphase: n = 86 cells, 3 coverslips; Mitotic: n = 75 cells, 3 coverslips. ‘*’ indicates statistically significant difference (t-test; p < 0.0001). Error bars represent S.E.M.

Article Snippet: Note that the mitotic eYFP-STIM1 band is only faintly visible with the ProSci antibody and the endogenous mitotic STIM1 band is not visible at all, despite comparable protein amounts with the asynchronous samples (based on the STIM1 N-terminus blot).

Techniques: Comparison, Fluorescence, Western Blot, Control, Molecular Weight, Glycoproteomics, Expressing

Journal: Nature cell biology

Article Title: Phosphorylation of STIM1 Underlies Suppression of Store-operated Calcium Entry During Mitosis

doi: 10.1038/ncb1995

Figure Lengend Snippet: a) Confocal imaging was carried out with eYFP-STIM1 expressing HeLa cells that were fixed and immunostained for α-tubulin and stained with DAPI. The upper panel shows a single image plane of an interphase cell. In the lower panel, a series of 128 planar images of a mitotic cell was deconvolved and a resultant image plane from near the center of the cell is shown. The mitotic cell was selected from an asynchronous population and was not nocodazole-arrested. In the merged images, eYFP-STIM1 is shown in red, α-tubulin is shown in green, and co-localization appears yellow. b and c) Confocal images of eYFP-STIM1 in live interphase or nocodazole-arrested mitotic HEK293 cells prior to (Stores Full) and following Ca2+ store depletion with thapsigargin (2 μM). Both equatorial (upper panels) and cortical (lower panels) slices of the same cells are shown. Scalebars are 5 μm.

Article Snippet: Note that the mitotic eYFP-STIM1 band is only faintly visible with the ProSci antibody and the endogenous mitotic STIM1 band is not visible at all, despite comparable protein amounts with the asynchronous samples (based on the STIM1 N-terminus blot).

Techniques: Imaging, Expressing, Staining

Journal: Nature cell biology

Article Title: Phosphorylation of STIM1 Underlies Suppression of Store-operated Calcium Entry During Mitosis

doi: 10.1038/ncb1995

Figure Lengend Snippet: a) Endogenous STIM1 was immunoprecipitated from asynchronous or mitotic HeLa cell lysates and analyzed by Western blot with the phosphospecific MPM-2 antibody (right panel). The blot was then stripped and re-probed with an anti-STIM1 antibody (left panel) to reveal total STIM1 amounts. Full scans of these blots are shown in Supplemental Figure 6c. b) eYFP-STIM1 or 482STOP were immunoprecipitated from asynchronous or nocodazole-arrested mitotic HeLa cell lysates with an anti-eYFP antibody and analyzed by Western blot with the MPM-2 antibody (right panel). The blot was then stripped and re-probed with the anti-eYFP antibody to reveal total protein amounts (left panel). Full scans of these blots are shown in Supplemental Figure 6d. Although most STIM1 phosphorylation experiments were carried out with HeLa cells (see Methods), we also observed recognition of eYFP-STIM1 by MPM-2 in mitotic HEK293 cells (Supplemental Fig. 4b). c and d) Confocal images of interphase and nocodazole-arrested mitotic HEK293 cells co-expressing eYFP-STIM1 and CFP-Orai1 (left panels) or 482STOP and CFP-Orai1 (right panels) prior to (Rest) and following Ca2+ store depletion with thapsigargin (Tg; 2 μM). In merged images, CFP-Orai1 is red, eYFP-STIM1 or 482STOP are green, and co-localization is yellow. Scalebars are 5 μm. Images are representative of greater than 5 cells for each condition.

Article Snippet: Note that the mitotic eYFP-STIM1 band is only faintly visible with the ProSci antibody and the endogenous mitotic STIM1 band is not visible at all, despite comparable protein amounts with the asynchronous samples (based on the STIM1 N-terminus blot).

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

Journal: Nature cell biology

Article Title: Phosphorylation of STIM1 Underlies Suppression of Store-operated Calcium Entry During Mitosis

doi: 10.1038/ncb1995

Figure Lengend Snippet: a) SOCE responses to restoration of 1.0 mM external Ca2+ following Ca2+ store depletion with thapsigargin (Tg; 2 μM) were measured in interphase (black trace) and nocodazole-arrested mitotic (gray trace) HEK293 cells co-expressing eYFP-STIM1 and Orai1 (S1+O1), and interphase (dark blue) and mitotic (light blue) cells co-expressing 482STOP and Orai1 (482STOP+O1). Each trace represents the average response of 20–30 cells from a single experiment. b) The peak increase in fluorescence ratio above baseline following Ca2+ add-back was calculated and averaged for each cell from experiments as described in panel a. Interphase eYFP-STIM1+Orai1: n = 79 cells, 3 coverslips; interphase 482STOP+Orai1: n = 66 cells, 3 coverslips; mitotic eYFP-STIM1+Orai1: n = 53 cells, 3 coverslips; mitotic 482STOP+Orai1: n = 47 cells, 3 coverslips. ‘*’ indicates statistically significant difference (one-way ANOVA followed by Tukey-Kramer; p < 0.05). Error bars represent S.E.M. Whole-cell Icrac currents were measured in interphase (c) and nocodazole-arrested mitotic (d) HEK293 cells co-expressing eYFP-STIM1 and Orai1 (S1+O1; black traces) and 482STOP and Orai1 (482STOP+O1; blue traces). The whole-cell condition was achieved in the presence of 2.0 mM external Ca2+, followed by switch to a divalent-free (DVF) external solution at 60 seconds. External Ca2+ was restored at 120 seconds, and 5 μM Gd3+ was added at 180 seconds to demonstrate full inhibition of Icrac currents. Current densities measured at −100 and +100 mV are plotted, and each trace represents the response of a single cell. Current-voltage relationships for the Ca2+ currents just prior to the switch to DVF and for the peak DVF currents are shown on the right. Peak leak-subtracted Ca2+ (e) and Na+ (f) currents measured at −100 mV for each cell were averaged and plotted. Interphase eYFP-STIM1+Orai1: n = 9 cells; interphase 482STOP+Orai1: n = 13 cells; mitotic eYFP-STIM1+Orai1: n = 11 cells; mitotic 482STOP+Orai1: n = 14 cells. ’*’ indicates statistically significant difference (t-test, p < 0.0001). Error bars represent S.E.M.

Article Snippet: Note that the mitotic eYFP-STIM1 band is only faintly visible with the ProSci antibody and the endogenous mitotic STIM1 band is not visible at all, despite comparable protein amounts with the asynchronous samples (based on the STIM1 N-terminus blot).

Techniques: Expressing, Fluorescence, Inhibition

Journal: Nature cell biology

Article Title: Phosphorylation of STIM1 Underlies Suppression of Store-operated Calcium Entry During Mitosis

doi: 10.1038/ncb1995

Figure Lengend Snippet: a) Total lysates from asynchronous or nocodazole-arrested mitotic HeLa cells expressing eYFP-STIM1 (S1) or S668A were analyzed by Western blot with the anti-STIM1 C-terminus antibody from ProSci (upper panel). The blot was stripped and re-probed with an anti-STIM1 N-terminus antibody (lower panel). Expressed eYFP-tagged and endogenous STIM1 bands are visible. Note that the mitotic eYFP-STIM1 band is only faintly visible with the ProSci antibody and the endogenous mitotic STIM1 band is not visible at all, despite comparable protein amounts with the asynchronous samples (based on the STIM1 N-terminus blot). The values below the blots represent the intensity of the ProSci eYFP-STIM1 or S668A bands as a percentage of the STIM1 N-terminus bands. All lanes were from the same blot, but additional lanes between the eYFP-STIM1 and S668A lanes were removed. Full scans of these blots are shown in Supplemental Figure 6e. b) Crude membrane fractions from wildtype or eYFP-STIM1 expressing HEK293 cells were treated with (+) or without (−) recombinant Cdk1/cyclin B and analyzed by Western blot with the ProSci STIM1 C-terminus antibody (upper panel). Note the diminished immunoreactivity of STIM1 from Cdk1/cyclin B-treated samples, indicative of STIM1 phosphorylation at S668. Blots were stripped and reprobed with the N-terminus STIM1 antibody to reveal total STIM1 in each sample. Full scans of these blots are shown in Supplemental Figure 6f. c) 570STOP containing the indicated S486A or S492A mutations were immunoprecipitated from nocodazole-arrested mitotic HEK293 cell lysates with an anti-eYFP antibody and analyzed by Western blot with the MPM-2 antibody (upper panel). The blot was then stripped and re-probed with the anti-eYFP antibody to reveal total protein amounts (lower panel). Full scans of these blots are shown in Supplemental Figure 6g. d) Confocal images of mitotic, thapsigargin-treated cells expressing eYFP-STIM1 and CFP-Orai1 (left) or S486A/S668A and CFP-Orai1 (right). eYFP-STIM1 and S486A/S668A are green and CFP-Orai1 is red; scalebar is 5 μm. e) SOCE responses were measured upon 1.0 mM Ca2+ restoration in nocodazole-arrested mitotic HEK293 cells co-expressing Orai1 with eYFP-STIM1 (black trace), S486A (blue trace), S668A (green trace), or S486A/S668A (red trace). Each trace represents the averaged response of 20–30 cells from a single experiment. For the bar graph in the right panel, the peak increase in fluorescence ratio above baseline following Ca2+ add-back was calculated and averaged for each cell from experiments carried out as described. For comparison, data with 482STOP from Figure 4b are also shown. eYFP-STIM1: n = 78 cells, 3 coverslips; S486A: n = 87 cells, 3 coverslips; S668A: n = 89 cells, 3 coverslips; S486A/S668A: n = 95 cells, 3 coverslips. ‘*’ indicates statistically significant difference compared to eYFP-STIM1 (one-way ANOVA followed by Tukey-Kramer; p < 0.05). Error bars represent S.E.M. f) Whole-cell Icrac currents were measured in mitotic eYFP-STIM1+Orai1 (black) and S486A/S668A+Orai1 cells (red). The whole-cell condition was achieved in the presence of 2.0 mM external Ca2+, followed by switch to a divalent-free (DVF) external solution at 60 seconds. External Ca2+ was restored at 120 seconds, and 5 μM Gd3+ was added at 180 seconds to demonstrate full inhibition of Icrac currents. Current densities measured at −100 and +100 mV are plotted, and each trace represents the response of a single cell. The bargraphs show average peak leak-subtracted Ca2+ and Na+ currents (including non-responding cells). Mitotic eYFP-STIM1+Orai1: n = 12 cells; mitotic S486A/S668A+Orai1: n = 13 cells. Error bars represent S.E.M.

Article Snippet: Note that the mitotic eYFP-STIM1 band is only faintly visible with the ProSci antibody and the endogenous mitotic STIM1 band is not visible at all, despite comparable protein amounts with the asynchronous samples (based on the STIM1 N-terminus blot).

Techniques: Expressing, Western Blot, Membrane, Recombinant, Phospho-proteomics, Immunoprecipitation, Fluorescence, Comparison, Inhibition

Journal: PLoS ONE

Article Title: Store-Operated Ca 2+ Entry Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

doi: 10.1371/journal.pone.0123432

Figure Lengend Snippet: A. STIM1 protein expression after RNA inference. EA.hy926 cells were transfected for 48 h with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. Cells were harvested and total protein was extracted and subjected to western blotting with anti-STIM1 antibodies, with anti-GAPDH antibodies as a loading control. STIM1 expression was quantified and analyzed statistically based on three independent experiments. Transfected cells were also stimulated with 200 ng/ml HMGB1 (B) or 1 μM TG (C), followed by the addition of 2 mM CaCl2. Intracellular calcium transients were measured using an Olympus FV1000 confocal microscope. Peak intracellular Ca2+ was quantified during intracellular release or extracellular Ca2+ influx. D. HMGB1-induced permeability was inhibited by STIM1 knockdown. EA.hy926 cells were plated in the upper part of transwell chambers until the formation of a tight monolayer, then transfected with STIM1 siRNA-1, siRNA-2 or control (scrambled) siRNA. HMGB1 200 ng/ml was added and cells were incubated for an additional 24 h. After incubation, endothelial permeability was assessed, as described above. E. Representative immunoblots showing that STIM1 knockdown inhibits Src activation. Transfected cells were treated with or without 200 ng/ml HMGB1 for 2 h. Cell lysates were analyzed by SDS-PAGE followed by western blotting using antibodies against phosphorylated Src and Src. Data are presented as mean ± SD of three independent experiments. *Indicates significant difference compared with the control group (P<0.05).

Article Snippet: Two siRNAs against STIM1 (STIM1 siRNA-1: 5’-GGCUCUGGAUACAGUGCUCTT-3’ and STIM1 siRNA-2: 5’-GAAGCUGCGCGAUGAGAUCTT-3’ ) and Two siRNAs against Orai1 (Orai1 siRNA-1: 5’-CGAGCACTCCATGCAGGCG-3’ and Orai1 siRNA-2: 5’-GAAGCUGCGCGAUGAGAUCTT-3’ ) were designed and obtained from Shanghai GenePharma Co, Ltd. (Shanghai, China) and were transfected into EA.hy926 cells using a Lipo2000 Transfection Kit (Carlsbad, CA, USA).

Techniques: Expressing, Transfection, Control, Western Blot, Microscopy, Permeability, Knockdown, Incubation, Activation Assay, SDS Page

Journal: International Journal of Oncology

Article Title: MicroRNA-488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3

doi: 10.3892/ijo.2018.4483

Figure Lengend Snippet: Primer sequences for reverse transcription-quantitative polymerase chain reaction.

Article Snippet: The miR-488 mimics (50 nM; 5′-UUGAAAGGCUAUUUCUUGGUC-3′), miR-488 inhibitors (100 nM; 5′-GACCAAGAAAUAGCCUUUCAA-3′), negative control (NC; 50 nM; 5′-UUCUCCGAACGUGUCACGUTT-3′), siRNA for AQP3 (50 nM; sti-AQP3 sense, 5′-GGGUCGUCACUCCUUUAAUTT-3′ and antisense, 5′-AUUAAAGGAGUGACGACCCTT-3) and sti-NC (50 nM) were synthesized and purified by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Sequencing

Journal: International Journal of Oncology

Article Title: MicroRNA-488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3

doi: 10.3892/ijo.2018.4483

Figure Lengend Snippet: Expression of AQP3 and miR-488 in OS tissues and cell lines. (A) RT-qPCR analysis of AQP3 expression in OS tissues and adjacent normal bone tissues (n=6). Transcript levels were normalized to GAPDH expression. (B) Relative AQP3 expression was analyzed by RT-qPCR in 5 OS cell lines was normalized to GAPDH (n=6). (C) RT-qPCR analysis of miR-488 expression in OS tissues and adjacent normal bone tissues. Transcript levels were normalized to U6. (D) Relative miR-488 expression was analyzed by RT-qPCR in 5 OS cell lines was normalized to U6 (n=6). (E) Pearson's correlation analysis of the relative expression levels of miR-488 and the relative AQP3 mRNA expression levels in OS tissues. All data are presented as the mean ± standard error of the mean. * P<0.05, ** P<0.01, *** P<0.001 vs. normal tissues or hFOB 1.19. AQP3, aquaporin 3; miR, microRNA; OS, osteosarcoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: The miR-488 mimics (50 nM; 5′-UUGAAAGGCUAUUUCUUGGUC-3′), miR-488 inhibitors (100 nM; 5′-GACCAAGAAAUAGCCUUUCAA-3′), negative control (NC; 50 nM; 5′-UUCUCCGAACGUGUCACGUTT-3′), siRNA for AQP3 (50 nM; sti-AQP3 sense, 5′-GGGUCGUCACUCCUUUAAUTT-3′ and antisense, 5′-AUUAAAGGAGUGACGACCCTT-3) and sti-NC (50 nM) were synthesized and purified by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: MicroRNA-488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3

doi: 10.3892/ijo.2018.4483

Figure Lengend Snippet: AQP3 was a direct target of miR-488. U2OS cells were transfected with miR-488 mimic or inhibitor for 48 h. (A) Schematic representation of AQP3 3′-UTRs exhibiting a putative miRNA target site. (B) The analysis of the relative luciferase activities of AQP3-WT and AQP3-MUT. (C) The protein and mRNA expression of AQP3 was determined by western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. All data are presented as the mean ± standard error of the mean, n=6. ## P<0.01 vs. miR-NC or antti-miR-NC. The inhibitor NC, antti-miR-NC; AQP3, aquaporin 3; WT, wild-type; MUT, mutant; UTR, untranslated region; NC, negative control.

Article Snippet: The miR-488 mimics (50 nM; 5′-UUGAAAGGCUAUUUCUUGGUC-3′), miR-488 inhibitors (100 nM; 5′-GACCAAGAAAUAGCCUUUCAA-3′), negative control (NC; 50 nM; 5′-UUCUCCGAACGUGUCACGUTT-3′), siRNA for AQP3 (50 nM; sti-AQP3 sense, 5′-GGGUCGUCACUCCUUUAAUTT-3′ and antisense, 5′-AUUAAAGGAGUGACGACCCTT-3) and sti-NC (50 nM) were synthesized and purified by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Transfection, Luciferase, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Mutagenesis, Negative Control

Journal: International Journal of Oncology

Article Title: MicroRNA-488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3

doi: 10.3892/ijo.2018.4483

Figure Lengend Snippet: The effects of AQP3-silencing on the proliferation, invasion and epithelial-mesenchymal transition of osteosarcoma cells. U2OS cells were trans-fected with sti-AQP3 or sti-NC. (A) The protein and mRNA expression of AQP3 was determined by western blot analysis and RT-qPCR, respectively. (B) Cell proliferation was assessed using a BrdU-ELISA assay. (C) The mRNA expression of PCNA, CDK4, cyclin D1 and p27 was determined by RT-qPCR. (D) Invasion was assessed using a Transwell assay. (E) Protein expression of MMP-2, MMP-9 and TIMP-1 was detected by western blot analysis. (F) The expression of E-cadherin, N-cadherin and Vimentin was determined by western blot analysis. All data are presented as the mean ± standard error of the mean, n=6. ## P<0.01 vs. sti-NC. AQP3, aquaporin 3; si, small interfering RNA; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; BrdU, bromodeoxyuridine; PCNA, proliferating cell nuclear antigen; CDK4, cyclin-dependent kinase 4; MMP, matrix metalloproteinase; TIMP, TIMP metallopeptidase inhibitor; OD, optical density.

Article Snippet: The miR-488 mimics (50 nM; 5′-UUGAAAGGCUAUUUCUUGGUC-3′), miR-488 inhibitors (100 nM; 5′-GACCAAGAAAUAGCCUUUCAA-3′), negative control (NC; 50 nM; 5′-UUCUCCGAACGUGUCACGUTT-3′), siRNA for AQP3 (50 nM; sti-AQP3 sense, 5′-GGGUCGUCACUCCUUUAAUTT-3′ and antisense, 5′-AUUAAAGGAGUGACGACCCTT-3) and sti-NC (50 nM) were synthesized and purified by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transwell Assay, Small Interfering RNA, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: MicroRNA-488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3

doi: 10.3892/ijo.2018.4483

Figure Lengend Snippet: Overexpression of AQP3 promotes cell proliferation, invasion and epithelial-mesenchymal transition in osteosarcoma cells. U2OS cells were transfected with pcDNA3.1 or pcDNA-AQP3 vector. (A) Cell proliferation was assessed using a BrdU-ELISA assay. (B) Invasion was assessed using a Transwell assay. (C) The expression of E-cadherin, N-cadherin and Vimentin was determined by western blot analysis. All data are presented as the mean ± standard error of the mean, n=6. # P<0.05, ## P<0.01 vs. pcDNA3.1. AQP3, aquaporin 3; BrdU, bromodeoxyuridine; OD, optical density.

Article Snippet: The miR-488 mimics (50 nM; 5′-UUGAAAGGCUAUUUCUUGGUC-3′), miR-488 inhibitors (100 nM; 5′-GACCAAGAAAUAGCCUUUCAA-3′), negative control (NC; 50 nM; 5′-UUCUCCGAACGUGUCACGUTT-3′), siRNA for AQP3 (50 nM; sti-AQP3 sense, 5′-GGGUCGUCACUCCUUUAAUTT-3′ and antisense, 5′-AUUAAAGGAGUGACGACCCTT-3) and sti-NC (50 nM) were synthesized and purified by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Transwell Assay, Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: MicroRNA-488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3

doi: 10.3892/ijo.2018.4483

Figure Lengend Snippet: Overexpression of AQP3 partially promotes cell proliferation, invasion and epithelial-mesenchymal transition in miR-488-overexpressing osteosarcoma cells. U2OS cells were transfected with either miR-488 mimic with or without pcDNA-AQP3 vector. (A) The protein and mRNA expression of AQP3 was determined by western blot analysis and RT-qPCR, respectively. (B) Cell proliferation was assessed using a BrdU-ELISA assay. (C) The mRNA expression of PCNA, CDK4, cyclin D1 and p27 was determined by RT-qPCR. (D) Invasion was assessed using a Transwell assay. (E) Protein expression of MMP-2, MMP-9 and TIMP-1 was detected by western blot analysis. (F) The expression of E-cadherin, N-cadherin and Vimentin was determined by western blot analysis. All data are presented as the mean ± standard error of the mean, n=6. ## P<0.01, ### P<0.001 vs. pcDNA + miR-448 mimic. AQP3, aquaporin 3; miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OD, optical density; BrdU, bromodeoxyuridine; PCNA, proliferating cell nuclear antigen; CDK4, cyclin-dependent kinase 4; MMP, matrix metalloproteinase; TIMP, TIMP metallopeptidase inhibitor.

Article Snippet: The miR-488 mimics (50 nM; 5′-UUGAAAGGCUAUUUCUUGGUC-3′), miR-488 inhibitors (100 nM; 5′-GACCAAGAAAUAGCCUUUCAA-3′), negative control (NC; 50 nM; 5′-UUCUCCGAACGUGUCACGUTT-3′), siRNA for AQP3 (50 nM; sti-AQP3 sense, 5′-GGGUCGUCACUCCUUUAAUTT-3′ and antisense, 5′-AUUAAAGGAGUGACGACCCTT-3) and sti-NC (50 nM) were synthesized and purified by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transwell Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: MicroRNA-488 inhibits proliferation, invasion and EMT in osteosarcoma cell lines by targeting aquaporin 3

doi: 10.3892/ijo.2018.4483

Figure Lengend Snippet: A schematic figure showing miR-488 regulation of AQP3 and subsequent changes in cell cycle progression, cell invasion and EMT. miR, micro RNA; AQP3, aquaporin 3; EMT, epithelial-mesenchymal transition.

Article Snippet: The miR-488 mimics (50 nM; 5′-UUGAAAGGCUAUUUCUUGGUC-3′), miR-488 inhibitors (100 nM; 5′-GACCAAGAAAUAGCCUUUCAA-3′), negative control (NC; 50 nM; 5′-UUCUCCGAACGUGUCACGUTT-3′), siRNA for AQP3 (50 nM; sti-AQP3 sense, 5′-GGGUCGUCACUCCUUUAAUTT-3′ and antisense, 5′-AUUAAAGGAGUGACGACCCTT-3) and sti-NC (50 nM) were synthesized and purified by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques:

Journal: Oncology Research

Article Title: STIL enhances the development of lung adenocarcinoma by regulating the glycolysis pathway

doi: 10.32604/or.2024.048562

Figure Lengend Snippet: Overexpression of STIL in LUAD. (A) The STIL gene expression level was evaluated using the GEPIA database; (B) STIL gene association with overall LUAD patient survival; (C) Cox risk analysis of LUAD patients; (D and E) the levels of STIL in tissues and cells were quantified via Western blot (T: tumor, N: normal). Compared to normal or 16HBE group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: RiboBio (Guangzhou, China) synthesized siRNA molecules targeting STIL (si- STIL -1 and si- STIL -2), E2 promoter binding factor 1-1 (E2F1-1), E2F1-2, and a negative control (si-NC).

Techniques: Over Expression, Gene Expression, Western Blot

Journal: Oncology Research

Article Title: STIL enhances the development of lung adenocarcinoma by regulating the glycolysis pathway

doi: 10.32604/or.2024.048562

Figure Lengend Snippet: Univariate Cox regression analysis of prognostic factors in TCGA-LUAD patients

Article Snippet: RiboBio (Guangzhou, China) synthesized siRNA molecules targeting STIL (si- STIL -1 and si- STIL -2), E2 promoter binding factor 1-1 (E2F1-1), E2F1-2, and a negative control (si-NC).

Techniques: Expressing

Journal: Oncology Research

Article Title: STIL enhances the development of lung adenocarcinoma by regulating the glycolysis pathway

doi: 10.32604/or.2024.048562

Figure Lengend Snippet: Knockdown of STIL suppressed cell proliferation and enhanced cell apoptosis. (A) STIL protein levels were determined by Western blot; cell viability, proliferation, and apoptosis were examined by CCK-8 (B), EdU (C), and flow cytometry (D), respectively. Compared to the si-NC group, * p < 0.05, ** p < 0.01.

Article Snippet: RiboBio (Guangzhou, China) synthesized siRNA molecules targeting STIL (si- STIL -1 and si- STIL -2), E2 promoter binding factor 1-1 (E2F1-1), E2F1-2, and a negative control (si-NC).

Techniques: Knockdown, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Oncology Research

Article Title: STIL enhances the development of lung adenocarcinoma by regulating the glycolysis pathway

doi: 10.32604/or.2024.048562

Figure Lengend Snippet: Knockdown of STIL restrained cell migration, invasion, and cycle progression. Invasion and migration of cells were analyzed using the wound-healing (A) and transwell (B) assays; (C) protein levels of N-Cadherin, E-Cadherin, and vimentin were measured by Western blot. (D) Cell cycle distribution was determined using flow cytometry. (E) Western blotting was used to quantify the levels of the cell cycle-associated proteins, cyclin-dependent kinase 4 (CDK4), cyclin E1 (CCNE1), and cyclin D1 (CCND1). Compared to the si-NC group, * p < 0.05, ** p < 0.01.

Article Snippet: RiboBio (Guangzhou, China) synthesized siRNA molecules targeting STIL (si- STIL -1 and si- STIL -2), E2 promoter binding factor 1-1 (E2F1-1), E2F1-2, and a negative control (si-NC).

Techniques: Knockdown, Migration, Western Blot, Flow Cytometry

Journal: Oncology Research

Article Title: STIL enhances the development of lung adenocarcinoma by regulating the glycolysis pathway

doi: 10.32604/or.2024.048562

Figure Lengend Snippet: Upregulation of E2F1 promoted STIL expression level. (A and B) GSEA analysis of STIL in LUAD using data from “ENCODE_TF_ChIP-seq_2015.txt”; (C) possible transcription factor binding sites of STIL are predicted by the JASPAR software; (D) analysis of the E2F1 gene expression level using the GEPIA database; (E) the association between the E2F1 gene and the overall survival rates of patients with LUAD; (F) correlation analysis of E2F1 and STIL ; dual-luciferase reporter assay (G) and ChIP assay (H) was used to verify the interaction of STIL and E2F1; (I) protein level of E2F1 and STIL were tested by Western blot. Compared to the pGL3-STIL-WT + Vector group, # p < 0.05. Compared to pGL3-STIL-WT + E2F1 or si-NC groups, * p < 0.05, ** p < 0.01.

Article Snippet: RiboBio (Guangzhou, China) synthesized siRNA molecules targeting STIL (si- STIL -1 and si- STIL -2), E2 promoter binding factor 1-1 (E2F1-1), E2F1-2, and a negative control (si-NC).

Techniques: Expressing, ChIP-sequencing, Binding Assay, Software, Gene Expression, Luciferase, Reporter Assay, Western Blot, Plasmid Preparation

Journal: Oncology Research

Article Title: STIL enhances the development of lung adenocarcinoma by regulating the glycolysis pathway

doi: 10.32604/or.2024.048562

Figure Lengend Snippet: Effects of E2F1 on cell viability and invasion ability were regulated by STIL . (A and B) protein levels of E2F1 and STIL were determined by Western blotting; cell viability and invasion were evaluated by CCK-8 (C) and transwell (D) assays. (E) Western blotting was used to measure the expression levels of the cell cycle-related proteins, CDK4, CCNE1, and CCND1. Compared to the si-NC + Vector group, * p < 0.05, ** p < 0.01. Compared to the si-E2F1-1 + Vector group; # p < 0.05, ## p < 0.01.

Article Snippet: RiboBio (Guangzhou, China) synthesized siRNA molecules targeting STIL (si- STIL -1 and si- STIL -2), E2 promoter binding factor 1-1 (E2F1-1), E2F1-2, and a negative control (si-NC).

Techniques: Western Blot, CCK-8 Assay, Expressing, Plasmid Preparation

Journal: Oncology Research

Article Title: STIL enhances the development of lung adenocarcinoma by regulating the glycolysis pathway

doi: 10.32604/or.2024.048562

Figure Lengend Snippet: Glycolysis regulated by STIL . (A and B) GSEA analysis of STIL in LUAD using data from “h.all.v2023.1.Hs.symbols.gmt”; (C–E) Glucose absorption, lactate production, and ATP/ADP ratio were assessed using kits; (F) Protein levels of HK-2, GLUT-1, and LDHA were assessed by Western blot. (G) ECAR and (H) OCR in LUAD cells after STIL knockdown. (I) The protein levels of HK-2, GLUT-1, and LDHA were tested by Western blot after treatment with Z-VAD-FMK. Compared with the si-NC group; * p < 0.05.

Article Snippet: RiboBio (Guangzhou, China) synthesized siRNA molecules targeting STIL (si- STIL -1 and si- STIL -2), E2 promoter binding factor 1-1 (E2F1-1), E2F1-2, and a negative control (si-NC).

Techniques: Western Blot, Knockdown

Journal: Journal of Ginseng Research

Article Title: The purified extract of steamed Panax ginseng protects cardiomyocyte from ischemic injury via caveolin-1 phosphorylation-mediating calcium influx

doi: 10.1016/j.jgr.2023.07.003

Figure Lengend Snippet: Primer Sequence of qRT-PCR

Article Snippet: Antibodies of GAPDH, and ORAI1, TRPC1 and STIM1 were purchased from Servicebio, and Boster Biological Technology, Wuhan, China, respectively.

Techniques: Sequencing