wtap overexpression plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: Post-transcriptional regulation of IFI16 promotes inflammatory endothelial pathophenotypes observed in pulmonary arterial hypertension

doi: 10.1101/2024.09.19.613988

Figure Lengend Snippet: ( A ) By RT-PCR, relative IFI16 mRNA was measured in lung tissue from PAH patients (n=4) vs. control (n=5). ( B ) By RT-PCR, relative IFI16 expression (n=3/group) was measured in IL-1β-treated (2 ng/mL) vs. vehicle control (0 ng/mL) PAECs (n=3/group). ( C ) By RT- PCR, relative IFI16 expression was measured following siRNA-mediated BMPR2 knockdown (siBMPR2) vs. control (siNC) PAECs (n=3/group). ( D ) By RT-PCR, relative IFI16 expression was measured in IFNΓ-treated vs. vehicle control PAECs (n=3/group). By RT-PCR, relative expression was of inflammatory cytokines ( E ) IL-6, ( F ) NF-κB, and ( G ) IL-1β were measured in IFI16- deficient (siIFI16) vs. control (siNC) PAECs (n=3/group) ( H-I ) By RT-PCR, relative expression was measured of endothelial inflammatory markers VCAM1 and ICAM1 in IFI16-deficient (siIFI16) vs. control (siNC) PAECs (n=3/group, H ); conversely, relative expression of these markers was measured following overexpression (LV-IFI16) vs. control (LV-Con) transduction IFI16 in PAECs (n=6/group, I ). ( J ) Relative caspase 3/7 activity was measured after IFI16 knockdown (siIFI16) vs control in PAECs +/- IL-1β (n=3/group). ( K ) Relative caspase 3/7 activity was measured following IFI16 overexpression (LV-IFI16) vs. control (LV-Con) transduction in PAECs (n=6/group). In all panels, mean expression in control groups was assigned a fold change of 1, to which relevant samples were compared. P-values calculated by two-tailed Student’s t test ( A-I, K ), and two-way ANOVA and post-hoc Bonferroni test ( J ), presented as mean +/- SEM.

Article Snippet: DNA plasmid of IFI16 (Addgene, Plasmid #53741) and WTAP (Addgene, Plasmid #35064) were purchased.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Knockdown, Over Expression, Transduction, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Post-transcriptional regulation of IFI16 promotes inflammatory endothelial pathophenotypes observed in pulmonary arterial hypertension

doi: 10.1101/2024.09.19.613988

Figure Lengend Snippet: ( A ) Schematic of DRACH motif (represented in yellow) of 5’UTR of IFI16 transcript. ( B ) Fold change of m6A/A was measured by colorimetric assay in IL-1β-treated vs. control (vehicle) PAECs (n=6/group). ( C ) A heatmap was generated showing differentially expressed m6A regulators in PAECs treated with or without IL-1β normalized to mean transcript levels in vehicle group, with *** representing p<0.001 and **** representing p<0.0001. ( D ) By RT- PCR, relative WTAP transcript expression was measured (n=3/group) in IL-1β-treated (2 ng/mL) vs. vehicle control (0 ng/mL) PAECs (n=3/group). ( E ) By RT-PCR, relative WTAP expression was measured following siRNA-mediated BMPR2 knockdown (siBMPR2) vs. control (siNC) PAECs (n=3/group). ( F ) Fold change of m6A/A was measured by colorimetric assay in WTAP-deficient (siWTAP) vs. control (siNC) PAECs (n=6/group). ( G ) Fold change of m6A/A was measured by colorimetric assay following WTAP overexpression (LV-WTAP) vs. control (LV-Con) PAECs (n=6/group). ( H ) By RT-PCR, relative IFI16 expression was measured in WTAP-deficient (siWTAP #1) vs. control (siNC) PAECs (n=6/group). ( I ) By RT-PCR, relative IFI16 expression was measured after WTAP overexpression (LV-WTAP) vs. control (LV-Con) transduction in PAECs (n=6/group). ( J ) By MeRIP pulldown (α-m6A vs. IgG control) and RT-PCR, fold enrichment of m6A methylation of IFI16 transcript following IL-1β treatment was measured in WTAP-deficient (siWTAP) vs. control (siNC) PAECs, normalized to IgG negative control. ( K ) IFI16 mRNA decay was measured in WTAP-deficient (siWTAP) vs. control (siNC) PAECs following inhibition of cellular transcription by actinomycin D (n=3/group, presented as mean and 95% CI). In B-J , mean expression in control groups was assigned a fold change of 1, to which relevant samples were compared. P-values calculated by two-tailed Student’s t test ( B, D-I ) and one-way ANOVA and post-hoc Bonferroni test ( J ), presented as mean +/- SEM.

Article Snippet: DNA plasmid of IFI16 (Addgene, Plasmid #53741) and WTAP (Addgene, Plasmid #35064) were purchased.

Techniques: Colorimetric Assay, Control, Generated, Reverse Transcription Polymerase Chain Reaction, Expressing, Knockdown, Over Expression, Transduction, Methylation, Negative Control, Inhibition, Two Tailed Test

Journal: bioRxiv

Article Title: Post-transcriptional regulation of IFI16 promotes inflammatory endothelial pathophenotypes observed in pulmonary arterial hypertension

doi: 10.1101/2024.09.19.613988

Figure Lengend Snippet: ( A ) Representative images of immunofluorescent staining of IFI16 (red), von Willebrand factor (vWF, endothelial marker, green), and DAPI (blue) in pulmonary vessels, scale bar: 50 µm in vehicle-treated vs. MCT-treated rats. ( B ) Relative IFI16 fluorescence co-localized with vWF- positive signal in vehicle-treated vs. MCT-treated rats. ( C ) Relative IFI16 quantified in total vessel in vehicle-treated vs. MCT-treated rats. ( D ) Representative images of immunofluorescent staining of WTAP (red), von Willebrand factor (vWF, endothelial marker, green), and DAPI (blue) in pulmonary vessels, scale bar: 50 µm in vehicle-treated vs. MCT-treated rats. ( E ) Relative WTAP fluorescence co-localized with vWF-positive signal in vehicle-treated vs. MCT-treated rats. ( F ) Relative WTAP quantified in total vessel in vehicle-treated vs. MCT-treated rats.

Article Snippet: DNA plasmid of IFI16 (Addgene, Plasmid #53741) and WTAP (Addgene, Plasmid #35064) were purchased.

Techniques: Staining, Marker, Fluorescence

Journal: bioRxiv

Article Title: Post-transcriptional regulation of IFI16 promotes inflammatory endothelial pathophenotypes observed in pulmonary arterial hypertension

doi: 10.1101/2024.09.19.613988

Figure Lengend Snippet: We demonstrate that IL-1β enhances WTAP activity and downstream methylation of IFI16 mRNA. The resulting up-regulation of IFI16 increases proinflammatory cytokine expression and apoptosis, with implications in PH pathogenesis. IL-1β, interleukin-1β; WTAP, Wilms’ tumor associated protein; and IFI16, interferon gamma inducible protein 16. Images generated by Biorender.com.

Article Snippet: DNA plasmid of IFI16 (Addgene, Plasmid #53741) and WTAP (Addgene, Plasmid #35064) were purchased.

Techniques: Activity Assay, Methylation, Expressing, Wilms Tumor Assay, Generated

Journal: PLoS Biology

Article Title: Dynamic assembly of the mRNA m 6 A methyltransferase complex is regulated by METTL3 phase separation

doi: 10.1371/journal.pbio.3001535

Figure Lengend Snippet: (A) Representative images showing localization of EGFP control, EGFP-METTL14 or EGFP-WTAP after Cry2-mCh-METTL3 condensates formation by light stimulation. Scale bar = 10 μm. (B) Colocalization of RNA (SYTO RNAselect live cell stain) and Cry2-mCh-METTL3 condensates. Cry2olig-mCh (Addgene #60032) is used as light-inducible oligomerization control. Scale bar = 10 μm. (C) Hypothetical models of intermolecular interactions mRNA m 6 A methyltransferase complex with (right) or without (left) self-interacting ability of METTL3. (D) Representative images showing localization of EGFP-METTL3 after Cry2-mCh-METTL3 condensates formation by light stimulation. (E) Co-immunoprecipitation assay with HEK293T coexpressing HA-METTL3 and FLAG-tagged METTL3 (M3), METTL14 (M14), or WTAP (WT). (F) Recruitment of METTL14 binding–deficient mutant METTL3 into Cry2-mCh-METTL3 liquid condensates. Scale bar = 10 μm. (G) Recruitment of METTL3 into Cyr2-mCh-METTL3 ΔLH mutant liquid condensates. Scale bar = 10 μm. LLPS, liquid–liquid phase separation; METTL3, methyltransferase-like 3; METTL14, methyltransferase-like 14; WTAP, Wilms tumor suppressor-1–associated protein.

Article Snippet: EGFP was N-terminally inserted into pcDNA3/FLAG METTL3, METTL14 and WTAP (Addgene #53741) for coexpression and FLIM experiments.

Techniques: Control, Staining, Co-Immunoprecipitation Assay, Binding Assay, Mutagenesis, Wilms Tumor Assay

Journal: PLoS Biology

Article Title: Dynamic assembly of the mRNA m 6 A methyltransferase complex is regulated by METTL3 phase separation

doi: 10.1371/journal.pbio.3001535

Figure Lengend Snippet: (A) (Top) In vivo EGFP lifetime maps of EGFP control, EGFP-METTL3, EGFP-METTL14, and EGFP-WTAP. Scale bar = 10 μm. (Bottom) Color heatmap representing mean fluorescent lifetime values (ns) measured from nucleoplasm without condensates (Nucleoplasm) or inside condensates (Condensates) of each EGFP-fused protein. (B) Histograms showing fluorescent lifetime distribution (>300,000 photons) measured from nucleoplasm without condensates (Nucleoplasm) or inside condensates (Condensates) of each EGFP-fused protein. # and vertical dashed line indicate control EGFP lifetime in nucleoplasm without condensates. (C) Schematic diagram describing the intermolecular distances and FRET activity based on observations. The underlying data for the graphs presented can be found in For Plots. FLIM-FRET, fluorescent lifetime imaging microscopy–Förster resonance energy transfer; METTL3, methyltransferase-like 3; METTL14, methyltransferase-like 14; WTAP, Wilms tumor suppressor-1–associated protein.

Article Snippet: EGFP was N-terminally inserted into pcDNA3/FLAG METTL3, METTL14 and WTAP (Addgene #53741) for coexpression and FLIM experiments.

Techniques: In Vivo, Control, Activity Assay, Imaging, Microscopy, Förster Resonance Energy Transfer, Wilms Tumor Assay

Journal: Cancer cell international

Article Title: Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression.

doi: 10.1186/s12935-021-02288-x

Figure Lengend Snippet: Fig. 3 Knockout of m6A writers downregulates UCA1 expression. A Me-RIP and qRT-PCR assays suggest the m6A modification of UCA1 in HCT-116 and DLD-1 cells. B and C Knockout of METTL3 impairs UCA1 expression in HCT-116 cells. B Representative western blot result showed knockout of METTL3 in HCT-116 cells. C downregulation of UCA1 in HCT-116 METTL3 KO cells was detected by qRT-PCR. D and E Knockout of METTL3 impairs UCA1 expression in DLD-1 cells. D, Representative western blot showing knockout of METTL3 in DLD-1 cells. E downregulation of UCA1 in DLD-1 METTL3 KO cells was detected by qRT-PCR. F and G Knockout of WTAP suppresses UCA1 expression in HCT-116 cells. F Representative western blot showing knockout of METTL3 in HCT-116 cells. G, downregulation of UCA1 in HCT-116 WTAP KO cells was detected by qRT-PCR. H and I Knockout of WTAP suppresses UCA1 expression in DLD-1 cells. H, Representative western blot showing knockout of WTAP in DLD-1 cells. I downregulation of UCA1 in DLD-1 WTAP KO cells was detected by qRT-PCR. Error bars represent SD, n = 3, **P < 0.01, ***P < 0.005

Article Snippet: WTAP (#53741) and METTL3 (#53739) expression vectors for rescue experiments were obtained from Addgene [19].

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Modification, Western Blot

Journal: Cancer cell international

Article Title: Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression.

doi: 10.1186/s12935-021-02288-x

Figure Lengend Snippet: Fig. 4 Re-expression of m6A writer restores the expression of UCA1 in KO cells. A–D Re-expression of METTL3 in KO cells restores UCA1 RNA level. A and C the representative western blot showing re-expression of METTL3 in HCT-116 KO clone#13 and DLD-1 KO clone#11. B and D the RNA level of UCA1 was detected by qRT-PCR. E–H Re-expression of WTAP in KO cells rescues UCA1 RNA level. E and G the representative western blot showing re-expression of WTAP in HCT-116 KO clone#28 and DLD-1 KO clone#37. F and H the RNA level of UCA1 was detected by qRT-PCR. Error bars represent SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.005

Article Snippet: WTAP (#53741) and METTL3 (#53739) expression vectors for rescue experiments were obtained from Addgene [19].

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Cancer cell international

Article Title: Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression.

doi: 10.1186/s12935-021-02288-x

Figure Lengend Snippet: Fig. 5 Knockout of METTL3 and WTAP impairs proliferation and survival of CRC cells. A METTL3 KO suppresses cell proliferation of HCT-116 cells, as determined by CCK-8 assay. B METTL3 KO inhibits colony formation of HCT-116 cells. Left, the representative picture of cell colonies; right, the statistical result. C and D Cell proliferation and colony formation assay in METTL3 KO DLD-1 cells. E KO of WTAP suppressed cell proliferation of HCT-116 cells. Cell proliferation was determined by CCK-8 assay. F WTAP KO inhibits colony formation of HCT-116 cells. Left, the representative picture of cell colonies; right, the statistical result. G and H Cell proliferation and colony formation assay in WTAP KO DLD-1 cells. Error bars represent SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.005

Article Snippet: WTAP (#53741) and METTL3 (#53739) expression vectors for rescue experiments were obtained from Addgene [19].

Techniques: Knock-Out, CCK-8 Assay, Colony Assay