dusp8  (Novus Biologicals)

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 1 Downregulation of DUSP8 in LUAD correlated with poor OS. A mRNA expression analysis of DUSP8, DUSP10, and DUSP16 in samples obtained from patients with LUAD (n = 513) compared to non-tumor tissues (NTs) (n = 287) from the GTEx–TCGA patient cohort. B The clinical outcome associated with DUSP8, DUSP10, and DUSP16 expression in LUAD using KM Plotter [36]. C Expression of MAPK8 and MAPK9 in LUAD (n = 513) vs. NT (n = 287) respectively. D Correlation between OS of patients with LUAD and expressions of MAPK8 and MAPK9. E Protein interaction network using STRING. The edges indicate both functional and physical protein associations, line thickness indicates the strength of data support. F Correlation between OS of female and male patients in all stages of LUAD and DUSP8 expression. G DUSP8 protein level measured using ELISA (n = 5). H Western blot analysis of DUSP8 expression in NTs and tumor samples from the same patient (n = 3). I Representative images of DUSP8 (brown) co-staining with CD45 or pan-CK (red), in LUAD and healthy donor tissue samples, (n = 3), scale bar 50 µm. Data are presented as mean ± standard error of the mean using a two-tailed unpaired t-test with Welch’s correction. For all analyses, P-values ≤0.05 were considered statistically significant. *p ≤0.05, **p ≤0.01, and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Functional Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 2 Upregulation of DUSP8 leads to a suppressive phenotype, whereas down-regulation of DUSP8 is tumor-promoting in vitro. Validation of DUSP8 overexpression after transfection of A549 cells with empty vector (EV) and DUSP8 expression vector (OE) was quantified using (A) qRT-PCR (n = 3), (B) immunofluoresence staining, and (C) western blot (n = 3). Phosphorylation of JNK was performed using a western blot of A549-EV and A549-DUSP8 OE cells (n = 3). D Comparison of colony formation between DUSP8 OE cells and EV control cells (n = 3). E Comparison of cellular proliferation between DUSP8 OE cells and EV control cells using BrdU assay (n = 3). F Migratory ability of DUSP8 OE cells assessed using Boyden chamber assay (n = 3). G Apoptosis of DUSP8 OE cells compared to EV control cells (n = 3). H Representative immunofluorescence images of EMT markers expression using antibodies against CK18 and VIM (green) counterstained with DAPI (blue), scale bars, 50 µm. I mRNA expression of DUSP8 after siRNA transfection with DUSP8 siRNA and non-targeting siRNA control (siNT) (n = 6). J Representative immunocytochemistry images of DUSP8 after treatment with DUSP8 siRNA compared to a non-targeting control. Cells in panels B and J were labeled using DUSP8 antibody and revealed by Alexa Fluor 488 secondary antibody (green). DNA was stained with DAPI (blue) (scale bars: 50 µm). K Western blotting of A549 cells after treatment with DUSP8 siRNA (n = 3). Functional assessment via L colony formation, M cell proliferation, N migration and O apoptosis of A549 cells after treatment with DUSP8 siRNA. P Representative immunofluorescence images of EMT marker expression using antibodies against CK18 and VIM (green) counterstained with DAPI (blue), scale bars, 50 µm. Data are shown as mean ± standard error of the mean using a two-tailed unpaired t-test with Welch’s correction. P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001 and ***p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vitro, Biomarker Discovery, Over Expression, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Staining, Western Blot, Phospho-proteomics, Comparison, Control, BrdU Staining, Boyden Chamber Assay, Immunocytochemistry, Labeling, Functional Assay, Migration, Marker, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 3 Impact of DUSP8 overexpression on cancer progression in vivo. A549-EV and A549-DUSP8 OE cells were injected into the right flank of immunodeficient mice. Tumors were harvested after 40 days. A Measurement of tumor size during tumor progression (n = 5). B Measurement of tumor mass after 40 days (n = 5). C Representative photographs of dissected DUSP8 overexpressing tumors, scale bar 5 mm. Validation of DUSP8 expression in mice tumor tissue samples via D mRNA (n = 5), E protein using ELISA (n = 5), and F western blot and G quantification of band intensity (n = 3). H Representative photomicrographs of TUNEL staining for apoptotic cells within the tumor counted per high power field (HPF) using Fiji Software (n = 5, 4 images per animal). I Representative photomicrographs of Ki67 staining of proliferating cells within the tumor counted per HPF using Fiji Software (n = 5, 5 images per animal). J Representative photomicrographs of vascular marker von Willebrand factor (vWF) quantified per HPF using Fiji (n = 5, 5 images per animal). Representative photomicrographs of EMT marker (K) CK18 and (L) VIM quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5, 3 images per animal). Ki67, vWF, CK18, and VIM staining were visualized using Alexa Flour 488 coupled secondary antibody (green). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (A) and two-tailed unpaired t-test with Welch’s correction (B–L). P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01 and **p ≤0.001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, In Vivo, Injection, Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, TUNEL Assay, Staining, Software, Marker, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 4 High expression of miR-147b promotes tumor progression in vitro and is correlated with low OS in lung cancer. A miR-147b - DUSP8 binding site predicted via RNA22. B Scatter plots of DUSP8 expression correlated with miR-147b expression in LUAD samples from the TCGA dataset (n = 34). The r-value and two-tailed, p-value were calculated using Pearson’s rank correlation coefficients. C In silico analysis of miR-147b expression in LUAD (n = 458) vs. non-tumor tissue (n = 46) from the same TCGA cohort. D Correlation between miR-147b expression and OS in patients with LUAD using Kaplan–Meier plotter. E Overexpression of miR-147b by transduced A549 cells compared to miR-SCR control (n = 3). F Luciferase reporter assay of 3’UTR DUSP8 transfected A549-miR-SCR vs. A549-miR-147b overexpressing cells (n = 3). G DUSP8 mRNA expression of A549-miR-147b OE vs. A549-miR-SCR transduced cells (n = 3). H Immunofluorescence staining of DUSP8 (green) counterstained with DAPI (blue), was quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5), scale bar 50 µm. I Western blot analysis of DUSP8 compared to loading control ACTB (n = 3). J Volcano Plot depicting DUSP8 as top downregulated gene upon miR-147b overexpression using NanoString. Assessment of K colony formation, L proliferation, M migration, and (N) apoptosis of A549-miR- 147b overexpressing cells compared to miR-SCR transduced cells (n = 3). Representative photomicrographs O of CK18 and P VIM antibody staining were visualized using Alexa Flour 488 coupled secondary antibody (green) and quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. P-values were determined using a two- tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, In Vitro, Binding Assay, Two Tailed Test, In Silico, Over Expression, Control, Luciferase, Reporter Assay, Transfection, Staining, Western Blot, Migration

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 5 Silencing miR-147b abrogates the oncogenic potential. Validation of miR-147b and DUSP8 after treatment of A549-miR-147b OE cells with miR-non-targeting control versus miR-147b inhibitor. A, B mRNA expression of miR-147b and DUSP8 (n = 6), C Immunocytochemistry staining of DUSP8 (green) counterstained with DAPI (blue), was quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5), scale bar 50 µm. D Western blot of DUSP8 compared to loading control ACTB (n = 3). Inhibition of miR-147b in functional assays in vitro performed via E apoptosis, F proliferation, G colony formation and H migration (n = 3). Representative photomicrographs I of CK18 and J VIM antibody staining were visualized using Alexa Flour 488 coupled secondary antibody (green) and quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. P-values were determined using a two-tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Biomarker Discovery, Control, Expressing, Immunocytochemistry, Staining, Western Blot, Inhibition, Functional Assay, In Vitro, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 6 High expression of DUSP8 in A549-miR-147b overexpressing cells rescues the tumor-promoting phenotype. A miR-147b expression in A549-miR-147b OE cells transfected with a DUSP8 overexpressing plasmid performed by qPCR (n = 3). Validation of DUSP8 in A549-miR- 147b-DUSP8 overexpressing cells at B mRNA level (n = 3) and C protein level, shown by immunocytochemistry staining of DUSP8 (green), counterstained with DAPI (blue), was quantified by calculating the mean fluorescence intensity (MFI) with Fiji (n = 5), scale bar 50 µm. D Western blot analysis of DUSP8 (n = 3). Quantification of E proliferation, F colony formation, G migration and H apoptosis of A549-miR-147b- DUSP8 overexpressing cells compared to A549-miR-147b transduced cells (n = 3). Representative photomicrographs of I CK18 and J VIM antibody staining were visualized with a secondary antibody (green) coupled to Alexa Flour 488. Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. Quantification of the mean fluorescence intensity of CK18 and VIM (n = 5). P-values were determined using a two- tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Biomarker Discovery, Immunocytochemistry, Staining, Western Blot, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 7 miR-147b overexpression leads to increased tumor burden in vivo. A Subcutaneous injection of A549-miR-SCR and A549-miR-147b cells into the right flank of immunodeficient NSG mice. Tumors were harvested after 40 days. A Representative macroscopic pictures of subcutaneous tumors, scale bar 5 mm. B Measurement of tumor size during tumor progression (n = 5) and C tumor mass after 40 days. Validation of D miR-147b and E DUSP8 mRNA expression in mice tumor samples (n = 5). F DUSP8 level measured by ELISA (n = 5). G Western blot analysis of DUSP8 in miR-147b OE tumors compared to miR-SCR tumors (n = 3). H Intravenous injection of A549-miR-SCR and A549-miR- 147b cells into the tail vein of immunodeficient NSG mice. Representative images of micro-CT scans, extracted lung images, H&E-stained sections, and immunofluorescence staining for Ki67 and vWF (green) and DAPI (blue) in tumor sections. Scale bars, 50 μm. I Quantification of average lung intensity (n = 5). Quantification of proliferating J Ki67+cells (green) (n = 5, 5 images per animal) and K vWF (n = 5, 5 images per animal) counterstained with DAPI (blue) within the tumor counted per high power field (HPF) using Fiji Software. Validation of L miR-147b and M DUSP8 mRNA expression in mice lung tumor samples (n = 5). N DUSP8 level measured by ELISA (n = 5). O Western blot analysis of DUSP8 expression (n = 3). P Schematic representation of miR-147b-mediated tumor progression via suppression of DUSP8. Suppression of DUSP8 by miR-147b leads to inhibition of JNK de-phosphorylation resulting in activation of JNK signaling. Overexpression of miR-147b alters the phosphorylation of MAPKs and leads to increased proliferation and migration. The opposite effects were observed by silencing miR-147b expression and thereby restoring DUSP8 function with cancer-suppressive properties, leading to increased apoptosis. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (B) and two-tailed unpaired t-test with Welch’s correction (C–O). P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001.

Article Snippet: The following primary antibodies were used: DUSP8 (1:1000, Novus Biologicals #31169, 1:1000, A9113 Antibodies.com, Stockholm, Sweden), mouse β-actin (1:5000, ab6276) from Abcam (Cambridge, UK), SAPK/JNK (1:1000 #9252), phospho-SAPK/JNK (1:1000, #4668), ASK1 (1:1000, #3762), phospho-ASK1 (1:1000, #3764), SEK1/MKK4 (1:1000, #9152), phospho-SEK1/MKK4 (1:1000, #9156), MKK7 (1:1000, #4172) and phospho-MKK7 (1:1000, #4171) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, In Vivo, Injection, Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Micro-CT, Staining, Software, Inhibition, De-Phosphorylation Assay, Activation Assay, Phospho-proteomics, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 1 Downregulation of DUSP8 in LUAD correlated with poor OS. A mRNA expression analysis of DUSP8, DUSP10, and DUSP16 in samples obtained from patients with LUAD (n = 513) compared to non-tumor tissues (NTs) (n = 287) from the GTEx–TCGA patient cohort. B The clinical outcome associated with DUSP8, DUSP10, and DUSP16 expression in LUAD using KM Plotter [36]. C Expression of MAPK8 and MAPK9 in LUAD (n = 513) vs. NT (n = 287) respectively. D Correlation between OS of patients with LUAD and expressions of MAPK8 and MAPK9. E Protein interaction network using STRING. The edges indicate both functional and physical protein associations, line thickness indicates the strength of data support. F Correlation between OS of female and male patients in all stages of LUAD and DUSP8 expression. G DUSP8 protein level measured using ELISA (n = 5). H Western blot analysis of DUSP8 expression in NTs and tumor samples from the same patient (n = 3). I Representative images of DUSP8 (brown) co-staining with CD45 or pan-CK (red), in LUAD and healthy donor tissue samples, (n = 3), scale bar 50 µm. Data are presented as mean ± standard error of the mean using a two-tailed unpaired t-test with Welch’s correction. For all analyses, P-values ≤0.05 were considered statistically significant. *p ≤0.05, **p ≤0.01, and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Expressing, Functional Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 2 Upregulation of DUSP8 leads to a suppressive phenotype, whereas down-regulation of DUSP8 is tumor-promoting in vitro. Validation of DUSP8 overexpression after transfection of A549 cells with empty vector (EV) and DUSP8 expression vector (OE) was quantified using (A) qRT-PCR (n = 3), (B) immunofluoresence staining, and (C) western blot (n = 3). Phosphorylation of JNK was performed using a western blot of A549-EV and A549-DUSP8 OE cells (n = 3). D Comparison of colony formation between DUSP8 OE cells and EV control cells (n = 3). E Comparison of cellular proliferation between DUSP8 OE cells and EV control cells using BrdU assay (n = 3). F Migratory ability of DUSP8 OE cells assessed using Boyden chamber assay (n = 3). G Apoptosis of DUSP8 OE cells compared to EV control cells (n = 3). H Representative immunofluorescence images of EMT markers expression using antibodies against CK18 and VIM (green) counterstained with DAPI (blue), scale bars, 50 µm. I mRNA expression of DUSP8 after siRNA transfection with DUSP8 siRNA and non-targeting siRNA control (siNT) (n = 6). J Representative immunocytochemistry images of DUSP8 after treatment with DUSP8 siRNA compared to a non-targeting control. Cells in panels B and J were labeled using DUSP8 antibody and revealed by Alexa Fluor 488 secondary antibody (green). DNA was stained with DAPI (blue) (scale bars: 50 µm). K Western blotting of A549 cells after treatment with DUSP8 siRNA (n = 3). Functional assessment via L colony formation, M cell proliferation, N migration and O apoptosis of A549 cells after treatment with DUSP8 siRNA. P Representative immunofluorescence images of EMT marker expression using antibodies against CK18 and VIM (green) counterstained with DAPI (blue), scale bars, 50 µm. Data are shown as mean ± standard error of the mean using a two-tailed unpaired t-test with Welch’s correction. P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001 and ***p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: In Vitro, Biomarker Discovery, Over Expression, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Staining, Western Blot, Phospho-proteomics, Comparison, Control, BrdU Staining, Boyden Chamber Assay, Immunocytochemistry, Labeling, Functional Assay, Migration, Marker, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 3 Impact of DUSP8 overexpression on cancer progression in vivo. A549-EV and A549-DUSP8 OE cells were injected into the right flank of immunodeficient mice. Tumors were harvested after 40 days. A Measurement of tumor size during tumor progression (n = 5). B Measurement of tumor mass after 40 days (n = 5). C Representative photographs of dissected DUSP8 overexpressing tumors, scale bar 5 mm. Validation of DUSP8 expression in mice tumor tissue samples via D mRNA (n = 5), E protein using ELISA (n = 5), and F western blot and G quantification of band intensity (n = 3). H Representative photomicrographs of TUNEL staining for apoptotic cells within the tumor counted per high power field (HPF) using Fiji Software (n = 5, 4 images per animal). I Representative photomicrographs of Ki67 staining of proliferating cells within the tumor counted per HPF using Fiji Software (n = 5, 5 images per animal). J Representative photomicrographs of vascular marker von Willebrand factor (vWF) quantified per HPF using Fiji (n = 5, 5 images per animal). Representative photomicrographs of EMT marker (K) CK18 and (L) VIM quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5, 3 images per animal). Ki67, vWF, CK18, and VIM staining were visualized using Alexa Flour 488 coupled secondary antibody (green). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (A) and two-tailed unpaired t-test with Welch’s correction (B–L). P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01 and **p ≤0.001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Injection, Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, TUNEL Assay, Staining, Software, Marker, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 4 High expression of miR-147b promotes tumor progression in vitro and is correlated with low OS in lung cancer. A miR-147b - DUSP8 binding site predicted via RNA22. B Scatter plots of DUSP8 expression correlated with miR-147b expression in LUAD samples from the TCGA dataset (n = 34). The r-value and two-tailed, p-value were calculated using Pearson’s rank correlation coefficients. C In silico analysis of miR-147b expression in LUAD (n = 458) vs. non-tumor tissue (n = 46) from the same TCGA cohort. D Correlation between miR-147b expression and OS in patients with LUAD using Kaplan–Meier plotter. E Overexpression of miR-147b by transduced A549 cells compared to miR-SCR control (n = 3). F Luciferase reporter assay of 3’UTR DUSP8 transfected A549-miR-SCR vs. A549-miR-147b overexpressing cells (n = 3). G DUSP8 mRNA expression of A549-miR-147b OE vs. A549-miR-SCR transduced cells (n = 3). H Immunofluorescence staining of DUSP8 (green) counterstained with DAPI (blue), was quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5), scale bar 50 µm. I Western blot analysis of DUSP8 compared to loading control ACTB (n = 3). J Volcano Plot depicting DUSP8 as top downregulated gene upon miR-147b overexpression using NanoString. Assessment of K colony formation, L proliferation, M migration, and (N) apoptosis of A549-miR- 147b overexpressing cells compared to miR-SCR transduced cells (n = 3). Representative photomicrographs O of CK18 and P VIM antibody staining were visualized using Alexa Flour 488 coupled secondary antibody (green) and quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. P-values were determined using a two- tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Binding Assay, Two Tailed Test, In Silico, Over Expression, Control, Luciferase, Reporter Assay, Transfection, Staining, Western Blot, Migration

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 5 Silencing miR-147b abrogates the oncogenic potential. Validation of miR-147b and DUSP8 after treatment of A549-miR-147b OE cells with miR-non-targeting control versus miR-147b inhibitor. A, B mRNA expression of miR-147b and DUSP8 (n = 6), C Immunocytochemistry staining of DUSP8 (green) counterstained with DAPI (blue), was quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5), scale bar 50 µm. D Western blot of DUSP8 compared to loading control ACTB (n = 3). Inhibition of miR-147b in functional assays in vitro performed via E apoptosis, F proliferation, G colony formation and H migration (n = 3). Representative photomicrographs I of CK18 and J VIM antibody staining were visualized using Alexa Flour 488 coupled secondary antibody (green) and quantified via calculation of the mean fluorescent intensity (MFI) using Fiji (n = 5). Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. P-values were determined using a two-tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Control, Expressing, Immunocytochemistry, Staining, Western Blot, Inhibition, Functional Assay, In Vitro, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 6 High expression of DUSP8 in A549-miR-147b overexpressing cells rescues the tumor-promoting phenotype. A miR-147b expression in A549-miR-147b OE cells transfected with a DUSP8 overexpressing plasmid performed by qPCR (n = 3). Validation of DUSP8 in A549-miR- 147b-DUSP8 overexpressing cells at B mRNA level (n = 3) and C protein level, shown by immunocytochemistry staining of DUSP8 (green), counterstained with DAPI (blue), was quantified by calculating the mean fluorescence intensity (MFI) with Fiji (n = 5), scale bar 50 µm. D Western blot analysis of DUSP8 (n = 3). Quantification of E proliferation, F colony formation, G migration and H apoptosis of A549-miR-147b- DUSP8 overexpressing cells compared to A549-miR-147b transduced cells (n = 3). Representative photomicrographs of I CK18 and J VIM antibody staining were visualized with a secondary antibody (green) coupled to Alexa Flour 488. Nuclear DNA was counterstained with DAPI (blue), scale bar 50 µm. Quantification of the mean fluorescence intensity of CK18 and VIM (n = 5). P-values were determined using a two- tailed unpaired t-test with Welsh’s correction. P-values ≤0.05 were considered statistically significant for all analyses. *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Plasmid Preparation, Biomarker Discovery, Immunocytochemistry, Staining, Western Blot, Migration, Two Tailed Test

Journal: Oncogene

Article Title: miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

doi: 10.1038/s41388-024-02969-7

Figure Lengend Snippet: Fig. 7 miR-147b overexpression leads to increased tumor burden in vivo. A Subcutaneous injection of A549-miR-SCR and A549-miR-147b cells into the right flank of immunodeficient NSG mice. Tumors were harvested after 40 days. A Representative macroscopic pictures of subcutaneous tumors, scale bar 5 mm. B Measurement of tumor size during tumor progression (n = 5) and C tumor mass after 40 days. Validation of D miR-147b and E DUSP8 mRNA expression in mice tumor samples (n = 5). F DUSP8 level measured by ELISA (n = 5). G Western blot analysis of DUSP8 in miR-147b OE tumors compared to miR-SCR tumors (n = 3). H Intravenous injection of A549-miR-SCR and A549-miR- 147b cells into the tail vein of immunodeficient NSG mice. Representative images of micro-CT scans, extracted lung images, H&E-stained sections, and immunofluorescence staining for Ki67 and vWF (green) and DAPI (blue) in tumor sections. Scale bars, 50 μm. I Quantification of average lung intensity (n = 5). Quantification of proliferating J Ki67+cells (green) (n = 5, 5 images per animal) and K vWF (n = 5, 5 images per animal) counterstained with DAPI (blue) within the tumor counted per high power field (HPF) using Fiji Software. Validation of L miR-147b and M DUSP8 mRNA expression in mice lung tumor samples (n = 5). N DUSP8 level measured by ELISA (n = 5). O Western blot analysis of DUSP8 expression (n = 3). P Schematic representation of miR-147b-mediated tumor progression via suppression of DUSP8. Suppression of DUSP8 by miR-147b leads to inhibition of JNK de-phosphorylation resulting in activation of JNK signaling. Overexpression of miR-147b alters the phosphorylation of MAPKs and leads to increased proliferation and migration. The opposite effects were observed by silencing miR-147b expression and thereby restoring DUSP8 function with cancer-suppressive properties, leading to increased apoptosis. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (B) and two-tailed unpaired t-test with Welch’s correction (C–O). P-values ≤0.05 were considered statistically significant for all analyses, *p ≤0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001.

Article Snippet: Sections were then blocked and incubated overnight with rabbit anti-DUSP8 antibody (1:1000; NBP1-88385) from Novus Biologicals (Centennial, CO, USA), CD45 monoclonal Antibody (CD45-2B11, Thermo Fisher, Waltham, MA, USA) and anti-pan cytokeratin antibody [C-11] (ab7753, Abcam, Cambridge, UK) as primary antibodies, followed by mmPRESS® Duet Double Staining Polymer Kit (HRP – anti-rabbit IgG-brown, AP – anti-mouse IgG-magenta) (MP-771415) (vector laboratories, Burlingame, CA, US) for detection according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Injection, Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Micro-CT, Staining, Software, Inhibition, De-Phosphorylation Assay, Activation Assay, Phospho-proteomics, Migration, Two Tailed Test

Journal: Stem Cells International

Article Title: Exosomal miR-423-5p Derived from Cerebrospinal Fluid Pulsation Stress-Stimulated Osteoblasts Improves Angiogenesis of Endothelial Cells via DUSP8/ERK1/2 Signaling Pathway

doi: 10.1155/2024/5512423

Figure Lengend Snippet: (a) bEnd.3 cells cocultured with OBs transfected with Cy3-labeled miR-423-5p mimic (red) in a transwell (0.4 μ m) plate; (b) representative images of OBs transfected with Cy3-miR-423-5p mimic; (c) the effects of GW4869 on exosome-dependent miRNA delivery from OBs into bEnd.3 cells; (d) the binging site for miRNA in DUSP8 mRNA; (e) a luciferase report assay; (f and g) western blotting. Data are represented as mean ± SD; n = 3. ∗ P < 0.05 vs. the Mimic, ∗∗ P < 0.01 vs. the Dusp8-WT NC, ∗∗∗ P < 0.001 vs. the Control, # P < 0.05 vs. the Inhibitor, ## P < 0.01 vs. the +OBs, ns P > 0.05 vs. Dusp8-WU NC.

Article Snippet: After 14 days, the nude mice were first euthanized by exposure to an increasing concentration of carbon dioxide; death was then confirmed by a physical process, and Matrigel plugs were collected, photographed, and performed for CD31 (1 : 100; Servicebio, China, Cat GB11063-2-100) immunofluorescence staining and DUSP8 (1 : 100; ABclonal, China, Cat A17990), pERK (1 : 100; Servicebio, China, Cat GB11507-100), and VEGF (1 : 100; Servicebio, China, Cat GB11034B-100) immunohistochemistry (IHC).

Techniques: Transfection, Labeling, Luciferase, Western Blot, Control

Journal: Stem Cells International

Article Title: Exosomal miR-423-5p Derived from Cerebrospinal Fluid Pulsation Stress-Stimulated Osteoblasts Improves Angiogenesis of Endothelial Cells via DUSP8/ERK1/2 Signaling Pathway

doi: 10.1155/2024/5512423

Figure Lengend Snippet: Exosomal miR-423-5p regulates angiogenesis in vivo: (a) image of Matrigel plugs after resection at 14 days; (b and c) western blot analysis of DUSP8 expression levels; (d, f, and g) immunohistochemical images analysis of p-ERK and VEGF-positive from Matrigel plugs; (e and h) images of CD31 detected by immunofluorescence staining in groups. ∗ P < 0.05 vs. the NC-Exos, ∗∗ P < 0.01 vs. the NC-Exos, ∗∗∗ P < 0.001 vs. the NC-Exos, ∗∗∗∗ P < 0.0001 vs. the NC-Exos.

Article Snippet: After 14 days, the nude mice were first euthanized by exposure to an increasing concentration of carbon dioxide; death was then confirmed by a physical process, and Matrigel plugs were collected, photographed, and performed for CD31 (1 : 100; Servicebio, China, Cat GB11063-2-100) immunofluorescence staining and DUSP8 (1 : 100; ABclonal, China, Cat A17990), pERK (1 : 100; Servicebio, China, Cat GB11507-100), and VEGF (1 : 100; Servicebio, China, Cat GB11034B-100) immunohistochemistry (IHC).

Techniques: In Vivo, Western Blot, Expressing, Immunohistochemical staining, Immunofluorescence, Staining

Journal: Stem Cells International

Article Title: Exosomal miR-423-5p Derived from Cerebrospinal Fluid Pulsation Stress-Stimulated Osteoblasts Improves Angiogenesis of Endothelial Cells via DUSP8/ERK1/2 Signaling Pathway

doi: 10.1155/2024/5512423

Figure Lengend Snippet: Model of exosomal miR-423-5p derived from CSFP-induced OBs in promoting angiogenesis of ECs. CSFP induces the secretion of OBs-derived exosomal miR-423-5p. OBs-derived exosomal miR-423-5p can directly target DUSP8 and then regulate the ERK1/2 signaling pathway to promote the angiogenesis of ECs.

Article Snippet: After 14 days, the nude mice were first euthanized by exposure to an increasing concentration of carbon dioxide; death was then confirmed by a physical process, and Matrigel plugs were collected, photographed, and performed for CD31 (1 : 100; Servicebio, China, Cat GB11063-2-100) immunofluorescence staining and DUSP8 (1 : 100; ABclonal, China, Cat A17990), pERK (1 : 100; Servicebio, China, Cat GB11507-100), and VEGF (1 : 100; Servicebio, China, Cat GB11034B-100) immunohistochemistry (IHC).

Techniques: Derivative Assay