Journal: Nature Communications

Article Title: EGFR/SRC/ERK-stabilized YTHDF2 promotes cholesterol dysregulation and invasive growth of glioblastoma

doi: 10.1038/s41467-020-20379-7

Figure Lengend Snippet: a Kaplan–Meier overall survival plot showing survival rates for lower-grade glioma and GBM patients having YTHDF2 low expression (blue), and high expression (red) in the TCGA PanCancer Atlas dataset ( http://www.cbioportal.org ) (two-sided log-rank test). b Kaplan–Meier overall survival plot showing survival rates for glioma patients having YTHDF2 low expression (blue), and high expression (red) in REMBRANDT dataset (two-sided log-rank test). c Kaplan–Meier overall survival plot showing survival rates for glioma patients having YTHDF2 low expression (blue), and high expression (red) in French dataset (two-sided log-rank test). d Profile of YTHDF2 mRNA expression in normal, GBM, or grade I–III glioma patients in TCGA and REMBRANDT datasets. e Analysis of YTHDF2 protein expression in 2 TMAs with 14 normal cores, 10 grade I astrocytomas cores, 47 grade II astrocytomas cores, 33 grade III astrocytomas cores, and 34 GBM cores; and in 36 GBM tissues, using immunohistochemical analysis. Scale bar = 100 μm. Representative image of two independent experiments. f Summary of YTHDF2 protein expression profile in ( e ). IHC staining score 0–1, low; 2–8, medium; 9–12, high (Kruskal–Wallis test and the post hoc Dunn’s multiple comparisons test). g Western blotting of YTHDF2 in glioma cells (Hs683, SW1783), GBM cells (T98G, U87 MG, U251 MG, LN229), and GSCs (GSC11, GSC17, GSC20, GSC23, GSC7-2, GSC6-27). Representative blot of two independent experiments. Source data are provided as a Source data file.

Article Snippet: Antibodies used in this study were the following: anti-LXRα (R&D Systems, Cat#PP-K8607-00, 1:500), anti-LXRβ (R&D Systems, Cat#PP-K8917-00, 1:500), anti-HIVEP2 (Invitrogen, Cat#PA5-100756, 1:500), anti-ERK1/2 (137F5) (Cell Signaling Technology, Cat#4695, 1:1000), anti-phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, Cat#4370, 1:2000), anti-EGFR (D38B1) (Cell Signaling Technology, Cat#4267, 1:1000), anti-phospho-EGFR (Y1173) (53A5) (Cell Signaling Technology, Cat#4407, 1:1000), anti-Src (Cell Signaling Technology, Cat#2109, 1:1000), anti-phospho-Src (Y419) (Invitrogen, Cat#44-660G, 1:800), anti-α-Tubulin (Santa Cruz Biotechnology, Cat#sc-5286, 1:1000), anti-BrdU (Cell Signaling Technology, Cat#5292, 1:200), anti-N 6 -methyladenosine (Synaptic Systems, Cat#202003), anti-YTHDF2 (Proteintech, Cat#24744-1-AP, 1:10,000), anti-YTHDF2 antibody for RIP (Aviva Systems Biology, ARP67917_P050), anti-FLAG (Sigma-Aldrich, Cat#F1804, 1:1000), anti-SOX2 (Cell Signaling Technology, Cat#3579, 1:1000), anti-GFAP (Cell Signaling Technology, Cat#3670, 1:1000), anti-Tuji-1 (Cell Signaling Technology, Cat#4466, 1:1000), anti-phosphoserine (Millipore, Cat#AB1603, 1:500), anti-phosphothreonine (Cell Signaling Technology, Cat#9386, 1:1000), anti-Ki-67 (Cell Signaling Technology, Cat#9664, 1:400), anti-cleaved caspase-3 (Cell Signaling Technology, Cat#9664, 1:1000), anti-rabbit IgG Alexa Fluor ® 488 (Invitrogen, Cat#A-11008, 1:1000), anti-mouse IgG Alexa Fluor ® 594 (Invitrogen, Cat#A-11001, 1:1000), anti-rabbit IgG HRP (Abcam, Cat#ab6721, 1:10,000), and anti-mouse IgG HRP (Santa Cruz, Cat# sc-516102, 1:5000).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Western Blot

Journal: Nature Communications

Article Title: EGFR/SRC/ERK-stabilized YTHDF2 promotes cholesterol dysregulation and invasive growth of glioblastoma

doi: 10.1038/s41467-020-20379-7

Figure Lengend Snippet: a Cell viability of shCtrl and shYTHDF2 GSC11 cells was measured. b Proliferation of shCtrl and shYTHDF2 GSC11 cells was assessed by 5-bromo-2’-deoxyuridine (BrdU) incorporation for 3 h. The percentage of BrdU positive cells was quantified. c In vitro invasion assay for shCtrl and shYTHDF2 GSC11 cells. Representative invasion images of the cells (left panel) and quantitation of relative cell invasion (right panel). Scale bar = 50 μm. d Nude mice intracranial tumor assay using shCtrl, shYTHDF2, and shYTHDF2 plus shRNA-resistant-YTHDF2 GSC11 cells. Brain sections stained with hematoxylin and eosin (H&E) show representative tumor xenografts. Tumor volumes were calculated. e , f Quantification of Ki-67 ( e ) or cleaved Caspase-3 ( f ) positive cells in brain tumor sections of shCtrl, shYTHDF2, and shYTHDF2 plus shRNA-resistant-YTHDF2 GSC11 cells. g In vivo invasion assay for shCtrl, shYTHDF2, and shYTHDF2 plus shRNA-resistant-YTHDF2 GSC11 cells. Representative H&E staining showing edges of the mice brain tumors (left panel), and the relative invasive fingers per tumor were counted (right panel). Scale bar = 200 μm. h Overall survival of mice injected with the indicated GSC11 cells. i Nude mice intracranial tumor assay using LN229 cells with vector only or with YTHDF2 overexpression. Brain sections stained with H&E show representative tumor xenografts. Tumor volumes were calculated. j In vivo invasion assay for LN229 cells with vector only or with YTHDF2 overexpression. Representative H&E staining showing edges of the mice brain tumors (left panel), and the relative invasive fingers per tumor were summarized (right panel). Scale bar = 200 μm. k Overall survival of mice injected with the indicated LN229 cells. For a – c , data are mean ± S.E.M., n = 3 biologically independent experiments (one-way ANOVA Tukey’s post hoc test). For d – k , n = 10 mice per group examined over two independent experiments. For d – g , data are mean ± S.D. (one-way ANOVA Tukey’s post hoc test). For h and k , two-sided log-rank test is conducted. For i and j , data are mean ± S.D. (unpaired two-sided t test). Source data are provided as a Source data file.

Article Snippet: Antibodies used in this study were the following: anti-LXRα (R&D Systems, Cat#PP-K8607-00, 1:500), anti-LXRβ (R&D Systems, Cat#PP-K8917-00, 1:500), anti-HIVEP2 (Invitrogen, Cat#PA5-100756, 1:500), anti-ERK1/2 (137F5) (Cell Signaling Technology, Cat#4695, 1:1000), anti-phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, Cat#4370, 1:2000), anti-EGFR (D38B1) (Cell Signaling Technology, Cat#4267, 1:1000), anti-phospho-EGFR (Y1173) (53A5) (Cell Signaling Technology, Cat#4407, 1:1000), anti-Src (Cell Signaling Technology, Cat#2109, 1:1000), anti-phospho-Src (Y419) (Invitrogen, Cat#44-660G, 1:800), anti-α-Tubulin (Santa Cruz Biotechnology, Cat#sc-5286, 1:1000), anti-BrdU (Cell Signaling Technology, Cat#5292, 1:200), anti-N 6 -methyladenosine (Synaptic Systems, Cat#202003), anti-YTHDF2 (Proteintech, Cat#24744-1-AP, 1:10,000), anti-YTHDF2 antibody for RIP (Aviva Systems Biology, ARP67917_P050), anti-FLAG (Sigma-Aldrich, Cat#F1804, 1:1000), anti-SOX2 (Cell Signaling Technology, Cat#3579, 1:1000), anti-GFAP (Cell Signaling Technology, Cat#3670, 1:1000), anti-Tuji-1 (Cell Signaling Technology, Cat#4466, 1:1000), anti-phosphoserine (Millipore, Cat#AB1603, 1:500), anti-phosphothreonine (Cell Signaling Technology, Cat#9386, 1:1000), anti-Ki-67 (Cell Signaling Technology, Cat#9664, 1:400), anti-cleaved caspase-3 (Cell Signaling Technology, Cat#9664, 1:1000), anti-rabbit IgG Alexa Fluor ® 488 (Invitrogen, Cat#A-11008, 1:1000), anti-mouse IgG Alexa Fluor ® 594 (Invitrogen, Cat#A-11001, 1:1000), anti-rabbit IgG HRP (Abcam, Cat#ab6721, 1:10,000), and anti-mouse IgG HRP (Santa Cruz, Cat# sc-516102, 1:5000).

Techniques: BrdU Incorporation Assay, In Vitro, Invasion Assay, Quantitation Assay, shRNA, Staining, In Vivo, Injection, Plasmid Preparation, Over Expression

Journal: Nature Communications

Article Title: EGFR/SRC/ERK-stabilized YTHDF2 promotes cholesterol dysregulation and invasive growth of glioblastoma

doi: 10.1038/s41467-020-20379-7

Figure Lengend Snippet: a Western blotting of YTHDF2 in GSC11 cells treated with inhibitors of AKT (MK-2206), FGFR (AZD4547), PDGFRα/β (CP673451), WNT (Wnt-C59), TGFβR1 (SB-431542), EGFR (Gefitinib), SRC (SU6656), and ERK1/2 (SCH772984) for 24 h. b Western blotting of YTHDF2 in U87/EGFR cells cultured with or without EGF. c Western blotting of YTHDF2 in LN229/EGFRvIII tet-on cells treated with increasing concentration of doxycycline (Dox, 0, 1, 10 μg/mL) for 48 h. d Stability of YTHDF2 was measured by cycloheximide (CHX, 50 μg/mL) chase assay in GSC11 cells with vehicle or Gefitinib treatment (left panel), and analysis of YTHDF2 protein lifetime (right panel). Data are mean ± S.D. of three independent experiments (unpaired two-sided t test). e Stability of YTHDF2 was measured by CHX (50 μg/mL) chase assay in LN229/EGFRvIII tet-on cells cultured with or without Dox (10 μg/mL, left panel), and analysis of YTHDF2 protein lifetime (right panel). Data are mean ± S.D. of three independent experiments (unpaired two-sided t test). f Western blotting of YTHDF2 in GSC11 cells cultured with or without EGF and treated with inhibitor of SRC (SU6656) or ERK1/2 (SCH772984). g Stability of YTHDF2 was measured by CHX chase assay in LN229/EGFRvIII tet-on cells cultured with Dox plus vehicle or SCH772984 treatment (left panel), and analysis of YTHDF2 protein lifetime (right panel). Data are mean ± S.D. of three independent experiments (unpaired two-sided t test). h Co-immunoprecipitation (co-IP) of YTHDF2 with phospho-ERK1/2 (Thr202/Tyr204) in whole-cell extracts from GSC11 cells cultured with or without EGF. i Co-IP of phospho-ERK1/2 (Thr202/Tyr204) with YTHDF2 in whole-cell extracts from GSC11 cells cultured with or without EGF. j Proximity ligation assay (PLA) of phospho-ERK1/2 and YTHDF2 in LN229/EGFRvIII tet-on or GSC11 cells (left panel) and quantification of average PLA puncta per cell (right panel). Scale bar = 20 μm. Data are mean ± S.D., n = 6 field pictures quantified over three independent experiments (unpaired two-sided t test). For a – c , f , h , and i , representative blots of three independent experiments are shown. Source data are provided as a Source data file.

Article Snippet: Antibodies used in this study were the following: anti-LXRα (R&D Systems, Cat#PP-K8607-00, 1:500), anti-LXRβ (R&D Systems, Cat#PP-K8917-00, 1:500), anti-HIVEP2 (Invitrogen, Cat#PA5-100756, 1:500), anti-ERK1/2 (137F5) (Cell Signaling Technology, Cat#4695, 1:1000), anti-phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, Cat#4370, 1:2000), anti-EGFR (D38B1) (Cell Signaling Technology, Cat#4267, 1:1000), anti-phospho-EGFR (Y1173) (53A5) (Cell Signaling Technology, Cat#4407, 1:1000), anti-Src (Cell Signaling Technology, Cat#2109, 1:1000), anti-phospho-Src (Y419) (Invitrogen, Cat#44-660G, 1:800), anti-α-Tubulin (Santa Cruz Biotechnology, Cat#sc-5286, 1:1000), anti-BrdU (Cell Signaling Technology, Cat#5292, 1:200), anti-N 6 -methyladenosine (Synaptic Systems, Cat#202003), anti-YTHDF2 (Proteintech, Cat#24744-1-AP, 1:10,000), anti-YTHDF2 antibody for RIP (Aviva Systems Biology, ARP67917_P050), anti-FLAG (Sigma-Aldrich, Cat#F1804, 1:1000), anti-SOX2 (Cell Signaling Technology, Cat#3579, 1:1000), anti-GFAP (Cell Signaling Technology, Cat#3670, 1:1000), anti-Tuji-1 (Cell Signaling Technology, Cat#4466, 1:1000), anti-phosphoserine (Millipore, Cat#AB1603, 1:500), anti-phosphothreonine (Cell Signaling Technology, Cat#9386, 1:1000), anti-Ki-67 (Cell Signaling Technology, Cat#9664, 1:400), anti-cleaved caspase-3 (Cell Signaling Technology, Cat#9664, 1:1000), anti-rabbit IgG Alexa Fluor ® 488 (Invitrogen, Cat#A-11008, 1:1000), anti-mouse IgG Alexa Fluor ® 594 (Invitrogen, Cat#A-11001, 1:1000), anti-rabbit IgG HRP (Abcam, Cat#ab6721, 1:10,000), and anti-mouse IgG HRP (Santa Cruz, Cat# sc-516102, 1:5000).

Techniques: Western Blot, Cell Culture, Concentration Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Proximity Ligation Assay

Journal: Nature Communications

Article Title: EGFR/SRC/ERK-stabilized YTHDF2 promotes cholesterol dysregulation and invasive growth of glioblastoma

doi: 10.1038/s41467-020-20379-7

Figure Lengend Snippet: a In vitro kinase assay of GST-YTHDF2. Recombinant active ERK1 and wild type (WT), S39A, T381A, or S39A/T381A YTHDF2 were incubated in the presence of [γ- 32 P] ATP. Proteins were separated by SDS-PAGE, transferred to PVDF membrane and autoradiography performed. MBP was included as positive control and co-incubated with SCH772984 as negative control. Coomassie blue stain was shown as loading control. Representative image of three independent experiments. b Stability of wild type or S39A/T381A mutated Flag-YTHDF2 was measured by CHX (50 μg/mL) chase assay in LN229/EGFRvIII cells cultured with Dox (10 μg/ml) (left panel) and analysis of YTHDF2 protein lifetime (right panel). Data are mean ± S.D. of three independent experiments. (unpaired two-sided t test). c In vitro invasion assay for LN229 cells transfected with wild type or S39A/T381A mutated Flag-YTHDF2. Representative invasion images of the cells (left panel) and quantitation of relative cell invasion (right panel). Data are mean ± S.E.M., n = 4 biologically independent experiments (one-way ANOVA Tukey’s post hoc test). Scale bar = 50 μm. d , e Cell viability of wild type or S39A/T381A mutated YTHDF2 expressing GSC11 ( e ) or GSC7-2 ( f ) cells with YTHDF2 depletion was measured by CCK-8. Data are mean ± S.E.M., n = 6 biologically independent experiments (one-way ANOVA Tukey’s post hoc test). f Nude mice intracranial tumor assay using wild type or S39A/T381A mutated YTHDF2 expressing GSC11 cells with YTHDF2 depletion. Brain sections stained with H&E show representative tumor xenografts. Tumor volumes were calculated using the formula V = ab 2 /2, where a and b are the tumor’s length and width, respectively. Data are mean ± S.D., n = 8 mice per group examined over two independent experiments (one-way ANOVA Tukey’s post hoc test). Source data are provided as a Source data file.

Article Snippet: Antibodies used in this study were the following: anti-LXRα (R&D Systems, Cat#PP-K8607-00, 1:500), anti-LXRβ (R&D Systems, Cat#PP-K8917-00, 1:500), anti-HIVEP2 (Invitrogen, Cat#PA5-100756, 1:500), anti-ERK1/2 (137F5) (Cell Signaling Technology, Cat#4695, 1:1000), anti-phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, Cat#4370, 1:2000), anti-EGFR (D38B1) (Cell Signaling Technology, Cat#4267, 1:1000), anti-phospho-EGFR (Y1173) (53A5) (Cell Signaling Technology, Cat#4407, 1:1000), anti-Src (Cell Signaling Technology, Cat#2109, 1:1000), anti-phospho-Src (Y419) (Invitrogen, Cat#44-660G, 1:800), anti-α-Tubulin (Santa Cruz Biotechnology, Cat#sc-5286, 1:1000), anti-BrdU (Cell Signaling Technology, Cat#5292, 1:200), anti-N 6 -methyladenosine (Synaptic Systems, Cat#202003), anti-YTHDF2 (Proteintech, Cat#24744-1-AP, 1:10,000), anti-YTHDF2 antibody for RIP (Aviva Systems Biology, ARP67917_P050), anti-FLAG (Sigma-Aldrich, Cat#F1804, 1:1000), anti-SOX2 (Cell Signaling Technology, Cat#3579, 1:1000), anti-GFAP (Cell Signaling Technology, Cat#3670, 1:1000), anti-Tuji-1 (Cell Signaling Technology, Cat#4466, 1:1000), anti-phosphoserine (Millipore, Cat#AB1603, 1:500), anti-phosphothreonine (Cell Signaling Technology, Cat#9386, 1:1000), anti-Ki-67 (Cell Signaling Technology, Cat#9664, 1:400), anti-cleaved caspase-3 (Cell Signaling Technology, Cat#9664, 1:1000), anti-rabbit IgG Alexa Fluor ® 488 (Invitrogen, Cat#A-11008, 1:1000), anti-mouse IgG Alexa Fluor ® 594 (Invitrogen, Cat#A-11001, 1:1000), anti-rabbit IgG HRP (Abcam, Cat#ab6721, 1:10,000), and anti-mouse IgG HRP (Santa Cruz, Cat# sc-516102, 1:5000).

Techniques: In Vitro, Kinase Assay, Recombinant, Incubation, SDS Page, Membrane, Autoradiography, Positive Control, Negative Control, Staining, Control, Cell Culture, Invasion Assay, Transfection, Quantitation Assay, Expressing, CCK-8 Assay

Journal: Nature Communications

Article Title: EGFR/SRC/ERK-stabilized YTHDF2 promotes cholesterol dysregulation and invasive growth of glioblastoma

doi: 10.1038/s41467-020-20379-7

Figure Lengend Snippet: a Bland-Altman plot of YTHDF2 RIP-seq in GSC11 cells. For each transcript, the average signal (measured as log 2 normalized reads counts) against the RIP-seq log 2 fold enrichment (RIP versus INPUT) was plotted. Significantly enriched targets are highlighted in red. b Volcano plot of RNA-seq results in siControl and siYTHDF2 GSC11 cells. Red dots indicate fold change >1.5 and p value <0.05. c Heatmap of expression and enrichment of 19 overlapped genes in RNA-seq, RIP-seq, and TCGA datasets results. d Schematic workflow of YTHDF2 downstream targets analysis. e , f Plots showing YTHDF2 binding to individual mRNAs of LXRA ( e ) and HIVEP2 ( f ) genes in GSC11 cells, as measured by RIP-seq. The normalized reads distribution for input (blue) and YTHDF2 RIP along mRNAs (red). g CLIP-qPCR showing the association of LXRA and HIVEP2 transcripts with YTHDF2 in GSC11 cells. Data are mean ± S.E.M., n = 4 biologically independent experiments (unpaired two-sided t test). h Analysis of mRNA levels of YTHDF2 , LXRA , HIVEP2 , and LXRB in GSC11 cells stably expressing control or YTHDF2 shRNA, using qPCR. Data are mean ± S.E.M., n = 3 biologically independent experiments (unpaired two-sided t test). i Analysis of mRNA levels of LXRA , HIVEP2 , and LXRB in LN229 cells transfected with vector or YTHDF2 expression constructs, using qPCR. Data are mean ± S.E.M., n = 5 biologically independent experiments (unpaired two-sided t test). j Kaplan–Meier overall survival plot showing survival rates for lower-grade glioma and GBM patients having both low LXRA expression (median) and high HIVEP2 expression (>median) (red) in the TCGA dataset (two-sided log-rank test). k Percentage of low LXRA / HIVEP2 expression and high LXRA / HIVEP2 expression in YTHDF2 low expression and high expression group. Chi-squared test, χ 2 = 69.63, p < 0.0001. Source data are provided as a Source data file.

Article Snippet: Antibodies used in this study were the following: anti-LXRα (R&D Systems, Cat#PP-K8607-00, 1:500), anti-LXRβ (R&D Systems, Cat#PP-K8917-00, 1:500), anti-HIVEP2 (Invitrogen, Cat#PA5-100756, 1:500), anti-ERK1/2 (137F5) (Cell Signaling Technology, Cat#4695, 1:1000), anti-phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, Cat#4370, 1:2000), anti-EGFR (D38B1) (Cell Signaling Technology, Cat#4267, 1:1000), anti-phospho-EGFR (Y1173) (53A5) (Cell Signaling Technology, Cat#4407, 1:1000), anti-Src (Cell Signaling Technology, Cat#2109, 1:1000), anti-phospho-Src (Y419) (Invitrogen, Cat#44-660G, 1:800), anti-α-Tubulin (Santa Cruz Biotechnology, Cat#sc-5286, 1:1000), anti-BrdU (Cell Signaling Technology, Cat#5292, 1:200), anti-N 6 -methyladenosine (Synaptic Systems, Cat#202003), anti-YTHDF2 (Proteintech, Cat#24744-1-AP, 1:10,000), anti-YTHDF2 antibody for RIP (Aviva Systems Biology, ARP67917_P050), anti-FLAG (Sigma-Aldrich, Cat#F1804, 1:1000), anti-SOX2 (Cell Signaling Technology, Cat#3579, 1:1000), anti-GFAP (Cell Signaling Technology, Cat#3670, 1:1000), anti-Tuji-1 (Cell Signaling Technology, Cat#4466, 1:1000), anti-phosphoserine (Millipore, Cat#AB1603, 1:500), anti-phosphothreonine (Cell Signaling Technology, Cat#9386, 1:1000), anti-Ki-67 (Cell Signaling Technology, Cat#9664, 1:400), anti-cleaved caspase-3 (Cell Signaling Technology, Cat#9664, 1:1000), anti-rabbit IgG Alexa Fluor ® 488 (Invitrogen, Cat#A-11008, 1:1000), anti-mouse IgG Alexa Fluor ® 594 (Invitrogen, Cat#A-11001, 1:1000), anti-rabbit IgG HRP (Abcam, Cat#ab6721, 1:10,000), and anti-mouse IgG HRP (Santa Cruz, Cat# sc-516102, 1:5000).

Techniques: RNA Sequencing, Expressing, Binding Assay, Stable Transfection, Control, shRNA, Transfection, Plasmid Preparation, Construct

Journal: Nature Communications

Article Title: EGFR/SRC/ERK-stabilized YTHDF2 promotes cholesterol dysregulation and invasive growth of glioblastoma

doi: 10.1038/s41467-020-20379-7

Figure Lengend Snippet: a Western blotting of LXRα and HIVEP2 in GSC11 cells stably transfected with control shRNA or two YTHDF2 shRNAs. b Western blotting of LXRα and HIVEP2 in LN229/EGFRvIII tet-on cells cultured with vehicle or Doxycycline and transfected with control siRNA or two individual YTHDF2 siRNAs. c , d MeRIP-seq of GSC11 cells shows m 6 A peaks at individual mRNAs of LXRA and HIVEP2 . The y -axis shows normalized reads distribution for input (blue) and m 6 A IP (red) along the transcripts. The schematic representation of mRNA of LXRA or HIVEP2 (top panel). e , f Lifetime of LXRA ( e ) and HIVEP2 ( f ) mRNA in GSC11 cells expressing shYTHDF2 (shDF2), or shControl (shCtrl). Transcription was inhibited by actinomycin D. g CLIP-qPCR showing the association of LXRA and HIVEP2 transcripts with wild-type (WT) or m 6 A recognition defective (MUT) Flag-YTHDF2 in LN229 cells. Data are mean ± S.E.M., n = 4 biologically independent experiments. h mRNA levels of YTHDF2 , LXRA , and HIVEP2 in LN229 cells transfected with vector, wild type (WT) or m 6 A recognition defective (MUT) YTHDF2. Data are mean ± S.E.M., n = 3 biologically independent experiments. i Western blotting of YTHDF2, LXRα, and HIVEP2 in LN229 cells transfected with vector, wild-type (WT) or recognition defective (MUT) YTHDF2. j , k Lifetime of LXRA ( j ) or HIVEP2 ( k ) mRNA in LN229 cells expressing wild-type (WT) or m 6 A recognition defective YTHDF2 (W432A/W486A/W491A, MUT). Transcription was inhibited by actinomycin D. l mRNA levels of LXRA and HIVEP2 in GSC11 cells treated with inhibitor of EGFR (Gefitinib), SRC (SU6656), or ERK1/2 (SCH772984). Data are mean ± S.E.M., n = 5 biologically independent experiments. m mRNA levels of LXRA and HIVEP2 in GSC11 cells with EGF intervention transfected with control or two individual YTHDF2 siRNAs. Data are mean ± S.E.M., n = 6 biologically independent experiments. n mRNA levels of LXRA and HIVEP2 in LN229/EGFRvIII tet-on cells cultured with or without Dox, and transfected with control or two individual YTHDF2 siRNAs. Data are mean ± S.E.M., n = 6 biologically independent experiments. For a , b , and i , representative blots of three independent experiments are shown. For g , h , and l – n , data were analyzed by one-way ANOVA Tukey’s post hoc test. Source data are provided as a Source data file.

Article Snippet: Antibodies used in this study were the following: anti-LXRα (R&D Systems, Cat#PP-K8607-00, 1:500), anti-LXRβ (R&D Systems, Cat#PP-K8917-00, 1:500), anti-HIVEP2 (Invitrogen, Cat#PA5-100756, 1:500), anti-ERK1/2 (137F5) (Cell Signaling Technology, Cat#4695, 1:1000), anti-phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, Cat#4370, 1:2000), anti-EGFR (D38B1) (Cell Signaling Technology, Cat#4267, 1:1000), anti-phospho-EGFR (Y1173) (53A5) (Cell Signaling Technology, Cat#4407, 1:1000), anti-Src (Cell Signaling Technology, Cat#2109, 1:1000), anti-phospho-Src (Y419) (Invitrogen, Cat#44-660G, 1:800), anti-α-Tubulin (Santa Cruz Biotechnology, Cat#sc-5286, 1:1000), anti-BrdU (Cell Signaling Technology, Cat#5292, 1:200), anti-N 6 -methyladenosine (Synaptic Systems, Cat#202003), anti-YTHDF2 (Proteintech, Cat#24744-1-AP, 1:10,000), anti-YTHDF2 antibody for RIP (Aviva Systems Biology, ARP67917_P050), anti-FLAG (Sigma-Aldrich, Cat#F1804, 1:1000), anti-SOX2 (Cell Signaling Technology, Cat#3579, 1:1000), anti-GFAP (Cell Signaling Technology, Cat#3670, 1:1000), anti-Tuji-1 (Cell Signaling Technology, Cat#4466, 1:1000), anti-phosphoserine (Millipore, Cat#AB1603, 1:500), anti-phosphothreonine (Cell Signaling Technology, Cat#9386, 1:1000), anti-Ki-67 (Cell Signaling Technology, Cat#9664, 1:400), anti-cleaved caspase-3 (Cell Signaling Technology, Cat#9664, 1:1000), anti-rabbit IgG Alexa Fluor ® 488 (Invitrogen, Cat#A-11008, 1:1000), anti-mouse IgG Alexa Fluor ® 594 (Invitrogen, Cat#A-11001, 1:1000), anti-rabbit IgG HRP (Abcam, Cat#ab6721, 1:10,000), and anti-mouse IgG HRP (Santa Cruz, Cat# sc-516102, 1:5000).

Techniques: Western Blot, Stable Transfection, Transfection, Control, shRNA, Cell Culture, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: EGFR/SRC/ERK-stabilized YTHDF2 promotes cholesterol dysregulation and invasive growth of glioblastoma

doi: 10.1038/s41467-020-20379-7

Figure Lengend Snippet: a Western blotting of YTHDF2, LXRα, and HIVEP2 in GSC11 cells stably expressing shCtrl, shYTHDF2, or shYTHDF2 plus shLXRA or/and shHIVEP2. Representative blot of three independent experiments is shown. b Proliferation of the cells in ( a ) was measured. Data are mean ± S.E.M., n = 6 biologically independent experiments. c In vitro invasion assay for GSC11 cells expressing shCtrl, shYTHDF2, or shYTHDF2 plus shLXRA or/and shHIVEP2. Data are mean ± S.E.M., n = 4 wells examined over three independent experiments. d , e mRNA levels of LXRA and its downstream targets in shCtrl and shYTHDF2 GSC11 cells ( d ) or in LN229 cells expressing vector or YTHDF2 plasmid ( e ). Data are mean ± S.E.M., n = 3 biologically independent experiments (unpaired two-sided t test). f , g Relative cellular cholesterol of GSC11 cells stably expressing shCtrl, shYTHDF2, shLXRA, or shYTHDF2 plus shLXRA ( f ) or LN229 cells expressing vector or YTHDF2 plasmid ( g ). Data are mean ± S.E.M., n = 6 ( f ) or n = 3 ( g ) biologically independent experiments. h , i Quantification of LDL uptake in GSC11 cells expressing shCtrl, shYTHDF2, or shYTHDF2 plus shLXRA ( h ) or in LN229 cells expressing wild-type (WT) or m 6 A recognition defective (MUT) YTHDF2 ( i ). Data are mean ± S.E.M., n = 3 biologically independent experiments. j In vitro invasion assay for GSC11 cells treated with conditioned culture medium from GSC11 cells transfected with siCtrl, siYTHDF2, siAPOE, or siYTHDF2 plus siAPOE. Data are mean ± S.E.M., n = 3 biologically independent experiments. k , l mRNA levels of HIVEP2 and its downstream target SSTR2 in shCtrl and shYTHDF2 GSC11 cells ( k ) or in LN229 cells expressing vector or YTHDF2 plasmid ( l ). Data are mean ± S.E.M., n = 3 ( k ) or n = 4 ( l ) biologically independent experiments (unpaired two-sided t test). m Nude mice intracranial tumor assay using shCtrl, shYTHDF2, and shYTHDF2 plus shLXRA and shHIVEP2 GSC11 cells. Brain sections stained with H&E show representative tumor xenografts. Tumor volumes were calculated. Data are mean ± S.D. n In vivo invasion assay for shCtrl, shYTHDF2, and shYTHDF2 plus shLXRA and shHIVEP2 GSC11 cells. Representative H&E staining showing edges of the mice brain tumors (left), and the relative invasive fingers per tumor were counted (right). Scale bar = 200 μm. Data are mean ± S.D. o Overall survival of mice injected with shCtrl, shYTHDF2, and shYTHDF2 plus shLXRA and shHIVEP2 GSC11 cells (two-sided log-rank test). For b , c , f , g , h , i , m , and n , data were analyzed by one-way ANOVA Tukey’s post hoc test. For m – o , n = 10 mice per group examined over two independent experiments. Source data are provided as a Source data file.

Article Snippet: Antibodies used in this study were the following: anti-LXRα (R&D Systems, Cat#PP-K8607-00, 1:500), anti-LXRβ (R&D Systems, Cat#PP-K8917-00, 1:500), anti-HIVEP2 (Invitrogen, Cat#PA5-100756, 1:500), anti-ERK1/2 (137F5) (Cell Signaling Technology, Cat#4695, 1:1000), anti-phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, Cat#4370, 1:2000), anti-EGFR (D38B1) (Cell Signaling Technology, Cat#4267, 1:1000), anti-phospho-EGFR (Y1173) (53A5) (Cell Signaling Technology, Cat#4407, 1:1000), anti-Src (Cell Signaling Technology, Cat#2109, 1:1000), anti-phospho-Src (Y419) (Invitrogen, Cat#44-660G, 1:800), anti-α-Tubulin (Santa Cruz Biotechnology, Cat#sc-5286, 1:1000), anti-BrdU (Cell Signaling Technology, Cat#5292, 1:200), anti-N 6 -methyladenosine (Synaptic Systems, Cat#202003), anti-YTHDF2 (Proteintech, Cat#24744-1-AP, 1:10,000), anti-YTHDF2 antibody for RIP (Aviva Systems Biology, ARP67917_P050), anti-FLAG (Sigma-Aldrich, Cat#F1804, 1:1000), anti-SOX2 (Cell Signaling Technology, Cat#3579, 1:1000), anti-GFAP (Cell Signaling Technology, Cat#3670, 1:1000), anti-Tuji-1 (Cell Signaling Technology, Cat#4466, 1:1000), anti-phosphoserine (Millipore, Cat#AB1603, 1:500), anti-phosphothreonine (Cell Signaling Technology, Cat#9386, 1:1000), anti-Ki-67 (Cell Signaling Technology, Cat#9664, 1:400), anti-cleaved caspase-3 (Cell Signaling Technology, Cat#9664, 1:1000), anti-rabbit IgG Alexa Fluor ® 488 (Invitrogen, Cat#A-11008, 1:1000), anti-mouse IgG Alexa Fluor ® 594 (Invitrogen, Cat#A-11001, 1:1000), anti-rabbit IgG HRP (Abcam, Cat#ab6721, 1:10,000), and anti-mouse IgG HRP (Santa Cruz, Cat# sc-516102, 1:5000).

Techniques: Western Blot, Stable Transfection, Expressing, In Vitro, Invasion Assay, Plasmid Preparation, Transfection, Staining, In Vivo, Injection

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: Analysis of the expression of YTHDF2 and its impact on the progression of NPC. (A) Comparison of the transcription levels of YTHDF2 in the combined GSE12452 , GSE180272 , GSE53819 , GSE61218 , and GSE64634 datasets. (B) Comparison of the expression level of YTHDF2 between malignant and nonmalignant cells in single-cell NPC dataset GSE150430 . (C) ROC curve analysis of YTHDF2 in diagnosing NPC. (D) Kaplan–Meier survival curve for YTHDF2 expression of NPC in GSE102349 .

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: Expressing, Comparison

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: The expression of FOXO1 in the transcription and translation of NPC cells and tissues. (A) Comparison of the transcription level of YTHDF2 in NPC cell lines with NP69. (B) Comparison of the transcription level of YTHDF2 in NPC tissues with Rhinitis.(A: n = 3 biological replicates, one-way ANOVA, B: t-test). (C-D) Comparison of the protein level of YTHDF2 in NPC cell lines with NP69. (E-F) Comparison of the protein level of YTHDF2 in NPC tissues with Rhinitis. (D: n = 3 biological replicates, one-way ANOVA, F: t-test). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns: not significant.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: Expressing, Comparison

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: In vitro functional experiments of YTHDF2 knockdown in NPC cells ( n = 3 biological replicates, student's t-test). (A-C) YTHDF2 knockdown inhibited the proliferation in 5-8F, CNE1 and HONE1cells. (D-G) YTHDF2 knockdown reduced the migration in 5-8F, CNE1 and HONE1 cells. (H-I) YTHDF2 knockdown reduced the invasive capabilities of 5-8F, CNE1 and HONE1 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: In Vitro, Functional Assay, Knockdown, Migration

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: In vitro functional experiments of YTHDF2 overexpression in NPC cells ( n = 3 biological replicates, student's t-test). (A-C) YTHDF2-transfected 5-8F, CNE1, and HONE1 cells exhibited accelerated proliferation. (D-G) Enhanced migratory capacity was observed in YTHDF2-overexpressing cells. (H-I) Matrigel invasion assays revealed significantly increased invasiveness following YTHDF2 overexpression. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: In Vitro, Functional Assay, Over Expression, Transfection

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: Correlation between YTHDF2 and FOXO1 expression ( n = 3 biological replicates, C-D: t-test, E-F: one-way ANOVA). (A) GSEA in GSE102349 revealed enrichment pathways in the YTHDF2-activated group. (B) Correlation analysis between YTHDF2 and FOXO1 expression in GSE102349 . Analysis of FOXO1 expression in 5-8F and CNE1 cells following YTHDF2 knockdown (C) or overexpression (D). Comparison of FOXO1 expression in NPC cell lines 5-8F (E) and CNE1 (F) which were treated with the m6A demethylase inhibitor 3-DAA for 48 h. * P < 0.05; ** P < 0.01; *** P < 0.001; ns: not significant.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: Expressing, Knockdown, Over Expression, Comparison

Journal: Cancer Biology & Therapy

Article Title: YTHDF2 enhances proliferation and metastasis of nasopharyngeal carcinoma by mediating m6A modification in destabilizing FOXO1 mRNA

doi: 10.1080/15384047.2025.2582349

Figure Lengend Snippet: YTHDF2 participates in m6A-mediated regulation of FOXO1. (A) Distribution of m6A modification sites of FOXO1 mRNA in 5-8F. (B) Volcano plot of MeRIP-seq analysis for YTHDF2. (C) RIP-qPCR exhibited the relative expression of FOXO1. (D) YFHDF2 binding interval in FOXO1. (E) M6A modification site on the 3'UTR of FOXO1.

Article Snippet: FOXO1 antibody (America, 2880) was procured from CST, and YTHDF2 antibody (China, Wuhan, Polyclonal Antibody for WB, IHC, IP, 24744−1-AP) and GAPDH antibody (China, Wuhan, Monoclonal Antibody for WB, IHC, 60004−1-Ig) were bought from Proteintech.

Techniques: Modification, Expressing, Binding Assay

Journal: Functional & Integrative Genomics

Article Title: ALKBH5-mediated NPC2 mRNA m 6 A demethylation promotes resistance to oxaliplatin in colorectal cancer

doi: 10.1007/s10142-025-01651-9

Figure Lengend Snippet: NPC2 was identified as a novel oxaliplatin resistance-related gene in CRC. ( A-B ) Venn diagram showing the co-upregulated and co-downregulated differentially expressed genes (DEGs) associated with oxaliplatin resistance between GSE76092 and GSE42387 . ( C ) Forest plot depicting the hazard ratio (HR) and p values for overall survival (OS) in patients with CRC based on high versus low expression of DEGs. ( D-F ) Kaplan-Meier analysis of OS, disease-specific survival (DSS) and progression-free interval (PFI) for CRC patients according to NPC2 expression based on Xiantao tool. ( G ) Kaplan-Meier analysis of OS for CRC patients according to NPC2 expression based on GEPIA2. ( H-I ) Kaplan-Meier analysis of OS and recurrence-free survival (RFS) for CRC patients according to NPC2 expression based on Kaplan-Meier plotter database. ( J ) Heatmap showing the relationship between NPC2 expression level and CRC sensitivity towards various therapeutic drugs based on BEST application. ( K ) Expression profiles of NPC2 in oxaliplatin treated and untreated cells based on the GSE47856 dataset. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001)

Article Snippet: The primary antibodies (Abs) against NPC2 (19888-1-AP)、GAPDH (60004-1-Ig)、YTHDF2 (24744-1-AP)、ALKBH5 (16837-1-AP)、FTO (27226-1-AP)、METLL3 (15073-1-AP) were purchased from Proteintech Group.

Techniques: Expressing

Journal: Functional & Integrative Genomics

Article Title: ALKBH5-mediated NPC2 mRNA m 6 A demethylation promotes resistance to oxaliplatin in colorectal cancer

doi: 10.1007/s10142-025-01651-9

Figure Lengend Snippet: NPC2 promotes the oxaliplatin resistance of CRC. ( A ) The oxaliplatin resistance of HCT116 and HCT116-OXR cells were detected by CCK-8 after treatment with the indicated concentrations of oxaliplatin for 48 h. ( B ) The colony formation assay was performed to determine the proliferation of HCT116 and HCT116-OXR cells after treatment with oxaliplatin (2 µg/mL). ( C ) Counting and statistical analysis of the colony numbers. ( D ) HCT116 cells were treated with oxaliplatin (5 µg/mL) for 48 h, the expression of NPC2 mRNA was measured by RT-qPCR. ( E ) the expression of NPC2 mRNA in HCT116 and HCT116-OXR cells were measured by RT-qPCR. ( F ) the expression of NPC2 protein in HCT116 and HCT116-OXR cells were measured by Western blot analysis. ( G , H ) HCT116 cells were transfected with NPC2-flag plasmid and the NPC2 level was measured by RT-qPCR ( G ) and Western blot analysis ( H ). ( I ) The oxaliplatin resistance of NPC2-overexpressed HCT116 cells was detected by CCK-8 after treatment with the indicated concentrations of oxaliplatin for 48 h. ( J ) HCT116 cells were transfected with control or NPC2 vector, and then treated with oxaliplatin (0.5 µg/mL), the proliferation of cells was determined by the colony formation assay. ( K ) Counting and statistical analysis of the colony numbers. ( L , M ) HCT116-OXR cells were transfected with NPC2-siRNA and the NPC2 level was measured by RT-qPCR ( L ) and Western blot analysis ( M ). ( N ) The oxaliplatin resistance of NPC2-silencing HCT116-OXR cells was detected by CCK-8 after treatment with the indicated concentrations of oxaliplatin for 48 h. ( O ) HCT116-OXR cells were transfected with control or NPC2-siRNA, and then treated with oxaliplatin (2 µg/mL), the proliferation of cells was determined by the colony formation assay. ( P ) Counting and statistical analysis of the colony numbers. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001)

Article Snippet: The primary antibodies (Abs) against NPC2 (19888-1-AP)、GAPDH (60004-1-Ig)、YTHDF2 (24744-1-AP)、ALKBH5 (16837-1-AP)、FTO (27226-1-AP)、METLL3 (15073-1-AP) were purchased from Proteintech Group.

Techniques: CCK-8 Assay, Colony Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Functional & Integrative Genomics

Article Title: ALKBH5-mediated NPC2 mRNA m 6 A demethylation promotes resistance to oxaliplatin in colorectal cancer

doi: 10.1007/s10142-025-01651-9

Figure Lengend Snippet: Oxaliplatin promotes NPC2 expression in an m 6 A dependent manner. ( A ) HCT116 and HCT116-OXR cells were treated with ActD (5 µg/mL) for the indicated times. The expression of NPC2 was examined with RT-qPCR. ( B ) HCT116 cells were treated with oxaliplatin (5 µg/mL) for 48 h, and the m 6 A modification levels of total RNA were detected by dot blot. ( C ) HCT116 cells were treated with oxaliplatin (5 µg/mL) for 48 h, the m 6 A modifications of NPC2 mRNA were measured by MeRIP-qPCR. ( D ) the m 6 A modifications of NPC2 mRNA in HCT116 and HCT116-OXR cells were measured by MeRIP-qPCR. ( E , F ) HCT116 cells were transfected with METTL3 plasmid, then the expression level of METTL3 and NPC2 were measured by RT-qPCR ( E ) and Western blot analysis ( F ). ( G , H ) HCT116 cells were transfected with NC or METTL3 siRNA, then the expression level of METTL3 and NPC2 were measured by RT-qPCR ( G ) and Western blot analysis ( H ). ( I , J ) HCT116 cells were transfected with FTO plasmid, then the expression level of FTO and NPC2 were measured by RT-qPCR ( I ) and Western blot analysis ( J ). ( K , L ) HCT116 cells were transfected with NC or FTO siRNA, then the expression level of FTO and NPC2 were measured by RT-qPCR ( K ) and Western blot analysis ( L ). ( M ) Schematic representation of pGL3-Basic-NPC2-WT and pGL3-Basic-NPC2-MUT promoter reporter plasmid. ( N ) HCT116 cells were co-transfected with pGL3-Basic-NPC2 promoter reporter plasmid and pRL-TK plasmid for 24 h, then the cells were treated with oxaliplatin (5 µg/mL) for 48 h. Relative luciferase activity was detected using a double luciferase reporter assay system ( n = 6). Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001)

Article Snippet: The primary antibodies (Abs) against NPC2 (19888-1-AP)、GAPDH (60004-1-Ig)、YTHDF2 (24744-1-AP)、ALKBH5 (16837-1-AP)、FTO (27226-1-AP)、METLL3 (15073-1-AP) were purchased from Proteintech Group.

Techniques: Expressing, Quantitative RT-PCR, Modification, Dot Blot, Transfection, Plasmid Preparation, Western Blot, Luciferase, Activity Assay, Reporter Assay

Journal: Functional & Integrative Genomics

Article Title: ALKBH5-mediated NPC2 mRNA m 6 A demethylation promotes resistance to oxaliplatin in colorectal cancer

doi: 10.1007/s10142-025-01651-9

Figure Lengend Snippet: ALKBH5 increases NPC2 expression in the oxaliplatin resistant CRC. ( A , B ) HCT116 and LoVo cells were treated with oxaliplatin (5 µg/mL) for 48 h, then the mRNA level of m 6 A regulators (METTL3, METTL14, METTL16, WATP, ALKBH5, FTO, IGF2BP1, IGF2BP2, IGF2BP3, YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2) were measured by RT-qPCR. ( C ) HCT116 cells were treated with oxaliplatin (5 µg/mL) for 48 h, the expression of NPC2 mRNA was measured by RT-qPCR. ( D ) HCT116 cells were treated with oxaliplatin (5 µg/mL) for 48 h, the protein expression of NPC2 and ALKBH5 was measured by Western blot analysis. ( E ) HCT116 cells were transfected with a luciferase reporter construct containing the ALKBH5 promoter, followed by treatment with oxaliplatin (5 µg/mL) for 48 h. Luciferase activity was measured and normalized to Renilla. ( F , G ) HCT116 cells were transfected with ALKBH5 plasmid, then the expression level of ALKBH5 and NPC2 were measured by RT-qPCR ( F ) and Western blot analysis ( G ). ( H ) HCT116 cells were transfected with ALKBH5 plasmid, and then treated with oxaliplatin (2 µg/mL), the proliferation of cells was determined by the colony formation assay. ( I ) Counting and statistical analysis of the colony numbers. ( J , K ) HCT116 cells were transfected with NC or ALKBH5 siRNA, then the expression level of ALKBH5 and NPC2 were measured by RT-qPCR ( J ) and Western blot analysis ( K ). ( L ) HCT116-OXR cells were transfected with control or ALKBH5-siRNA, and then treated with oxaliplatin (2 µg/mL), the proliferation of cells was determined by the colony formation assay. ( M ) Counting and statistical analysis of the colony numbers. ( N ) HCT116 cells were transfected with ALKBH5 plasmid, the m 6 A modifications of NPC2 mRNA were measured by MeRIP-qPCR. ( O ) HCT116 cells were transfected with ALKBH5 siRNA, the m 6 A modifications of NPC2 mRNA were measured by MeRIP-qPCR. ( P ) HCT116-OXR cells were treated with oxaliplatin (5 µg/mL), and RIP-PCR was performed to detect the molecular interaction within ALKBH5 and NPC2. ( Q ) The binding capacity between NPC2 mRNA and ALKBH5 protein or YTHDF2 protein in HCT116 cells were examined by RNA pulldown and western blot analysis. ( R ) HCT116 cells were transfected with ALKBH5 plasmid or ALKBH5 siRNA, then treated with ActD (5 µg/mL) for the indicated times. The expression of NPC2 mRNA was examined with RT-qPCR. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001)

Article Snippet: The primary antibodies (Abs) against NPC2 (19888-1-AP)、GAPDH (60004-1-Ig)、YTHDF2 (24744-1-AP)、ALKBH5 (16837-1-AP)、FTO (27226-1-AP)、METLL3 (15073-1-AP) were purchased from Proteintech Group.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Colony Assay, Control, Binding Assay

Journal: Functional & Integrative Genomics

Article Title: ALKBH5-mediated NPC2 mRNA m 6 A demethylation promotes resistance to oxaliplatin in colorectal cancer

doi: 10.1007/s10142-025-01651-9

Figure Lengend Snippet: YTHDF2 promotes the degradation of NPC2 mRNA in the oxaliplatin resistant CRC. ( A - E ) HCT116 cells were transfected with siRNA of YTHDF1 ( A ), YTHDF2 ( B ), YTHDF3 ( C ), YTHDC1 ( D ), YTHDC2 ( E ), then the mRNA level of YTH domain-containing proteins and NPC2 were measured by RT-qPCR. ( F ) HCT116 cells were transfected with NC or YTHDF2 siRNA, then the protein level of YTHDF2 and NPC2 were measured by Western blot analysis. ( G ) HCT116 cells were transfected with control or YTHDF2-siRNA, and then treated with oxaliplatin (2 µg/mL), the proliferation of cells was determined by the colony formation assay. ( H ) Counting and statistical analysis of the colony numbers. ( I ) HCT116-OXR cells were treated with oxaliplatin (5 µg/mL), and RIP-PCR was performed to detect the molecular interaction within YTHDF2 and NPC2. ( J ) HCT116 cells were transfected with NC or YTHDF2 siRNA, then treated with ActD (5 µg/mL) for the indicated times. The expression of NPC2 mRNA was examined with RT-qPCR. ( K ) HCT116 cells were transfected with NC or YTHDF2-siRNA then with or without ALKBH5 knockdown. The protein level of ALKBH5, YTHDF2 and NPC2 were measured by Western blot analysis. ( L ) HCT116 cells were transfected with Vector or YTHDF2 plasmid then with or without ALKBH5 overexpression. The protein level of ALKBH5, YTHDF2 and NPC2 were measured by Western blot analysis. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001)

Article Snippet: The primary antibodies (Abs) against NPC2 (19888-1-AP)、GAPDH (60004-1-Ig)、YTHDF2 (24744-1-AP)、ALKBH5 (16837-1-AP)、FTO (27226-1-AP)、METLL3 (15073-1-AP) were purchased from Proteintech Group.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Control, Colony Assay, Expressing, Knockdown, Plasmid Preparation, Over Expression

Journal: Functional & Integrative Genomics

Article Title: ALKBH5-mediated NPC2 mRNA m 6 A demethylation promotes resistance to oxaliplatin in colorectal cancer

doi: 10.1007/s10142-025-01651-9

Figure Lengend Snippet: ALKBH5/NPC2 knockdown reverses oxaliplatin resistance in vivo. ( A ) HCT116-OXR cells (4 × 10 6 cells) transfected with siNPC2 or siALKBH5 were subcutaneously xenografted into nude mice and tumors were allowed to develop for 10 days, then intraperitoneally injected with oxaliplatin (5 mg/kg/d/mouse) or DMSO every 2 days for 2 weeks. The tumor growth of subcutaneous xenografts in nude mice was monitored every 5 days and mice were sacrificed on Day 32. ( B-D ) The image ( B ), volume ( C ) and the weights ( D ) of subcutaneous xenografts from nude mice. Mice injected intraperitoneally with oxaliplatin (5 mg/kg/day per mouse) or DMSO 14 days after HCT116-OXR-siNC or siNPC2 cells plantation. ( E , F ) RT-qPCR ( E ) and Western blot assays ( F ) for the expression of NPC2 in each group of tumors from ( B ). ( G-I ) The image ( G ), volume ( H ) and the weights ( I ) of subcutaneous xenografts from nude mice. Mice injected intraperitoneally with oxaliplatin (5 mg/kg/day per mouse) or DMSO 14 days after HCT116-OXR-siNC or siALKBH5 cells plantation. ( J , K ) RT-qPCR ( J ) and Western blot assays ( K ) for the expression of ALKBH5 and NPC2 in each group of tumors from ( G ). Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001)

Article Snippet: The primary antibodies (Abs) against NPC2 (19888-1-AP)、GAPDH (60004-1-Ig)、YTHDF2 (24744-1-AP)、ALKBH5 (16837-1-AP)、FTO (27226-1-AP)、METLL3 (15073-1-AP) were purchased from Proteintech Group.

Techniques: Knockdown, In Vivo, Transfection, Injection, Quantitative RT-PCR, Western Blot, Expressing