Journal: Science Advances

Article Title: Regulation of telomere homeostasis and genomic stability in cancer by N 6 -adenosine methylation (m 6 A)

doi: 10.1126/sciadv.abg7073

Figure Lengend Snippet: ( A ) Heatmaps for differential m 6 A patterns between RWPE-1 and LNCaP. FE, fold enrichment of m 6 A signals over input; FC, fold change of m 6 A signals in LNCaP versus in RWPE-1 (L vs. R). ( B ) Volcano plot of differentially expressed genes upon knockdown of METTL3 in LNCaP cells. N , numbers of significantly changed genes [log 2 fold change (FC) = ±log 2 (1.3) indicated by the red dashed lines and P = 0.05 by the black dashed line]. ( C ) Venn diagram showing the overlap between differentially expressed genes upon METTL3 knockdown (shM3 versus shCtrl) and mRNAs with stronger m 6 A intensity in LNCaP cells (LNCaP cluster). ( D ) Scatterplot showing the expression changes of the overlapped genes in (C) in the METTL3 rescue system. ( E ) IGV browser tracks of MeRIP/m 6 A-seq data at the genomic location of HMBOX1 . ( F ) MeRIP-qPCR analysis of m 6 A signals on HMBOX1 mRNAs in the specified cells, which were infected with control shRNA (shCtrl) or shRNA targeting METTL3 (shM3#1). Normal rabbit IgG was included as the negative control. ( G and H ) Expression of METTL3 and HMBOX1 in the control (shCtrl) and METTL3 knockdown (shM3#1 and shM3#2) cells established in Huh-7 (G) and A549 (H). ( I and J ) mRNA (I) and protein (J) levels of METTL3 and HMBOX1 in the 22Rv1 xenograft model of prostate cancer. P values in (B) were calculated by the Wald test with Benjamini-Hochberg adjustment.

Article Snippet: Antibodies used in this study include αm 6 A (Millipore, ABE572 or MABE1006) for MeRIP/m 6 A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH.

Techniques: Knockdown, Expressing, Infection, Control, shRNA, Negative Control

Journal: Science Advances

Article Title: Regulation of telomere homeostasis and genomic stability in cancer by N 6 -adenosine methylation (m 6 A)

doi: 10.1126/sciadv.abg7073

Figure Lengend Snippet: ( A ) Levels of specified mRNA molecules in LNCaP cells transfected with control siRNA (siCtrl) or siRNA targeting METTL3 (siM3) for 72 hours. ( B and C ) Half-life of HMBOX1 mRNA in LNCaP cells upon silencing METTL3 (B) or ALKBH5 (C) with the treatment of ActD (5 μg/ml) before total mRNAs were collected at indicated time points. ( D ) Levels of indicated molecules upon YTHDF2 knockdown in LNCaP. ( E ) Expression of HMBOX1 in the control (siCtrl) and YTHDF2-knockdown (siDF2) cells of LNCaP stably expressing empty backbone (vector), wild-type METTL3 (M3-WT), or catalytically dead mutant (M3-CD). ( F ) Schematic illustration of effective (A) and noneffective (A-10nt) sgRNA targeting HMBOX1 transcript (sg HMBOX1 ). ( G ) MeRIP-qPCR analysis of m 6 A signals on HMBOX1 in LNCaP cells expressing dCas9 fused to the wild-type ALKBH5 (dCas9-ALKBH5-WT) or to the incompetent demethylase (dCas9-ALKBH5-H204A) together with control sgRNA (sgCtrl), sg HMBOX1 -A, or sg HMBOX1 -A-10nt. GAPDH was included as a negative control. ( H ) Expression of HMBOX1 in the CRISPR-dCas9–based, m 6 A-editing system that is described in (G). ( I ) Half-life of HMBOX1 mRNA in LNCaP cells expressing dCas9-ALKBH5-WT together with specified sgRNAs and treated with ActD as described in (B) and (C).

Article Snippet: Antibodies used in this study include αm 6 A (Millipore, ABE572 or MABE1006) for MeRIP/m 6 A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH.

Techniques: Transfection, Control, Knockdown, Expressing, Stable Transfection, Plasmid Preparation, Mutagenesis, Negative Control, CRISPR

Journal: Science Advances

Article Title: Regulation of telomere homeostasis and genomic stability in cancer by N 6 -adenosine methylation (m 6 A)

doi: 10.1126/sciadv.abg7073

Figure Lengend Snippet: ( A and B ) Relative telomere length measurement by qPCR in the specified LNCaP (A) and A549 (B) cells. EPC (LPC), early (late) passage cells. ( C and D ) Representative images (C) and quantitative analysis (D) of metaphase FISH in the specified A549 cells at late passages. Telomere was detected by TelG-Cy3 (red), and DNA was stained with DAPI (blue). Arrows, shortened telomeres; circle, eroded telomeres; arrowhead, lost telomeres. Scale bars, 10 μm. ( E and F ) IP in LNCaP upon knockdown of HMBOX1 (shHM1#1 and #2) (E) or stably expressing the indicated plasmid DNAs (F). ( G ) Representative images and quantification of TERC RNA FISH combined with TRF2 immunostaining in the specified cells. At least 60 nuclei were counted in each biological replicate to quantify the colocalization. ( H ) MeRIP-qPCR analysis of m 6 A intensities on HMBOX1 in LNCaP cells expressing dCas13b proteins with a nuclear localization signal (nls) fused to either the wild-type METTL3 (dCas13b-M3 WT nls) or the catalytically dead mutant (dCas13b-M3 CD nls), together with control sgRNA (sgCtrl) or sg HMBOX1 -A. ( I and J ) Expression of METTL3 and HMBOX1 (I) or IP (J) in dCas13b-based, m 6 A-editing system that is described in (H). ( K and L ) Representative images (K) and quantification (L) of the TIF assays in A549 cells with either low (EPC) or high (LPC) passage numbers. Cells with five or more colocalization of γH2AX foci and telomeres were scored as TIF positive. Asterisks in (E), (F), and (J), heavy chain of IgG.

Article Snippet: Antibodies used in this study include αm 6 A (Millipore, ABE572 or MABE1006) for MeRIP/m 6 A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH.

Techniques: Staining, Knockdown, Stable Transfection, Expressing, Plasmid Preparation, Immunostaining, Mutagenesis, Control

Journal: Science Advances

Article Title: Regulation of telomere homeostasis and genomic stability in cancer by N 6 -adenosine methylation (m 6 A)

doi: 10.1126/sciadv.abg7073

Figure Lengend Snippet: ( A and B ) Immunoblotting analysis in LNCaP (A) and A549 (B) expressing the indicated plasmid DNAs. EPC (LPC), early (late) passage cells. ( C and D ) Expression of TP53 , CDKN1A , and MDM2 by RT-qPCR (C) and recruitment of HMBOX1 to the promoter region of MDM2 (P1, P2, and P3) by ChIP-qPCR (D) in LNCaP cells as described in (A) and (B). ( E to G ) Expression of the indicated mRNA molecules (E), immunoblotting (F), and HMBOX1 binding at the indicated chromatin regions (G) in the dCas13b-based, m 6 A-editing system of LNCaP cells. ( H and I ) Representative images (H) and quantitative analysis (I) of CO-FISH in A549 cells at high passage numbers, which stably express the indicated plasmid DNAs. Telomere was detected by TelG-Cy3 (red), and DNA was stained with DAPI (blue). Scale bars, 10 μm. ( J and K ) Representative images (J) and quantification (K) of anaphase bridge (arrowhead) formation in late-passage A549 cells as described in (H) and (I). DNA was stained with DAPI (blue). Scale bars, 10 μm. Eight random microscopic views were examined in each biological replicate for quantification purpose.

Article Snippet: Antibodies used in this study include αm 6 A (Millipore, ABE572 or MABE1006) for MeRIP/m 6 A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH.

Techniques: Western Blot, Expressing, Plasmid Preparation, Quantitative RT-PCR, ChIP-qPCR, Binding Assay, Stable Transfection, Staining

Journal: Science Advances

Article Title: Regulation of telomere homeostasis and genomic stability in cancer by N 6 -adenosine methylation (m 6 A)

doi: 10.1126/sciadv.abg7073

Figure Lengend Snippet: ( A ) HMBOX1 expression in a panel of human tumors and the corresponding normal tissues in TCGA. Cancers that show significant down-regulation of HMBOX1 were highlighted in blue. ( B to D ) Comparison of HMBOX1 expression between tumors and the corresponding normal tissue counterparts in prostate (B) , liver (C) , and lung (D) cancer. N , case numbers. ( E and F ) MeRIP-qPCR analysis of m 6 A signals on HMBOX1 (E) or RT-qPCR detecting the expression of HMBOX1 (F) in 12 pairs of prostate cancer (PCa) and adjacent normal tissues (Normal). ( G ) Correlation of m 6 A signals on HMBOX1 with expression of METTL3 or HMBOX1 in the tumor tissues mentioned in (E) and (F). ( H ) Correlation between telomere length and expression of HMBOX1 or METTL3 in prostate tumors. ( I ) Correlation matrix showing correlation among the mRNA levels of METTL3 and HMBOX1 and fractions of altered genome in prostate cancer. Numbers in blue, correlation coefficients. Numbers in black, P values. ( J and K ) Correlation between the fractions of cancer genome with alterations and expression of METTL3 or HMBOX1 in liver (J) and lung (K) cancer. Correlation coefficients (Cor) were determined by Pearson correlation, and P values in (E) and (F) were calculated by two-tailed paired t test.

Article Snippet: Antibodies used in this study include αm 6 A (Millipore, ABE572 or MABE1006) for MeRIP/m 6 A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Two Tailed Test

Journal: Science Advances

Article Title: Regulation of telomere homeostasis and genomic stability in cancer by N 6 -adenosine methylation (m 6 A)

doi: 10.1126/sciadv.abg7073

Figure Lengend Snippet: Overexpression of METTL3 in human cancer leads to augmented m 6 A signals on HMBOX1 . Alteration to this particular m 6 A epitranscriptomic program facilitates the degradation of HMBOX1 mRNAs, causes progressive telomere shortening, inactivates p53 signaling, and eventually generates genomic instability in cancer cells, which drives the full malignant progression.

Article Snippet: Antibodies used in this study include αm 6 A (Millipore, ABE572 or MABE1006) for MeRIP/m 6 A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH.

Techniques: Over Expression