Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , b , Average histone modification levels, as well as input signals, in WT and Tet2 -KO mESCs at DNA hypermethylated regions ( a ) or hypomethylated regions ( b ), detected in Tet2 -KO mESCs. c , Average ATAC signals in WT, Tet2 -KO, and Pspc1 -KO mESCs at DNA 5mC hypermethylated regions or hypomethylated regions detected in Tet2 -KO mESCs. d , Correlation between DNA methylation differences in Tet2 -KO versus WT mESCs and changes in their downstream gene transcription. carRNAs were categorized into different groups, including eRNA, paRNA, and repeat RNA. Within each group of carRNAs, they were further divided into 50 bins based on the ranked DNA methylation differences upon Tet2 KO in mESCs. Error band represents standard errors. e , Schematics showing the dual mode of chromatin regulation by TET2 when bound to different protein partners. f , Overlapping ratios of regions marked by different histone modifications with DNA hypermethylated regions (detected in the Tet2 -KO mESCs). g , H2AK119ub and H3K27me3 chromatin bindings at IAP, MERVL, and LINE loci measured through CUT&Tag protocol. Comparisons against “0 h” group. h - i , Total number of ATAC-seq peaks in mESCs. h , Number of ATAC-seq peaks marked with H2AK119ub modification in mESCs; i , ATAC-seq peaks were categorized into four groups: differentially upregulated (Up), downregulated (Down), or unchanged (Invar) after Tet2 KO in mESCs. Additionally, they were classified based on their association with H2AK119ub as either marked (H2AK119ub) or not marked (notH2AK119ub). j , Volcano plot displaying changes in H2AK119ub peak intensity for Tet2 -KO versus WT mESCs. k , Overlap of ATAC-seq peaks with increased chromatin accessibility (up ATAC) or decreased H2AK119ub (Down H2AK119ub) in Tet2 -KO mESCs. l , Genomic distribution of H2AK119ub peaks in WT and Tet2 -KO mESCs. Line centre ( g ) represents mean and whiskers s.d. P values, Wald test ( j ), two-tailed unpaired t test with Welch’s correction ( g ), two-tailed t-distribution with n-2 degrees of freedom ( d ). NS, P > 0.05. n = 3 biological replicates ( g ). The depicted genome-wide data represent an integration of three biological replicates ( c , d , f , h - l ) or two biological replicates ( a , b ).

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Modification, DNA Methylation Assay, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , b , Feature enrichment in hypermethylated differentially methylated regions (DMRs) compared with hypomethylated DMRs (hyperspecific; a ), and in hypomethylated DMRs compared with hypermethylated DMRs (hypospecific; b ) using the odds ratio. c , Overlapping ratios of histone modifications with DNA hypomethylated regions for Tet2 -KO versus WT mES cells. d , The H2AK119ub levels at IAP loci after TET2-CD (catalytic domain; WT or catalytically dead HxD) tethering by dCas13. e , Representative image showing nucleic acids cross-linked to MBD6. f , Spike-in-calibrated ATAC–seq signals or H2AK119ub signals in WT (siNC WT) and Tet2- KO (siNC Tet2 KO) mES cells, as well as in Mbd6- KD Tet2 -KO (siMbd6 Tet2 KO) mES cells. g , Changes in H2AK119ub on m 5 C hypomethylated (hypo.), unchanged (invar.) and hypermethylated (hyper.) repeat RNA after Tet2 KO versus WT. h , H2AK119ub changes in m 5 C hypermethylated repeat RNA in Tet2 -KO versus WT compared with Mbd6- KD Tet2 -KO versus Tet2 -KO mES cells. i , The correlation between ATAC signal fold changes in Tet2 -KO versus WT mES cells, and comparing Mbd6 KD with control Tet2 -KO mES cells. PCC, Pearson correlation coefficient. j , The correlation between changes in ATAC and H2AK119ub signals, comparing Mbd6 KD with control Tet2- KO mES cells. k , The H2AK119ub levels and ATAC signal at IAP RNA after dCas13–MBD6-MBD tethering. l , Schematics of the proposed pathway of MBD6 regulating chromatin state through caRNA m 5 C binding. Data are mean ± s.d. ( d and k ) and mean ± s.e.m. ( f ). For g and h , the box plots show the median (centre line), the upper and lower quartiles (box limits) and 1.5× the interquartile range (whiskers). P values were calculated using two-tailed unpaired t -tests with Welch’s correction ( d and k ), two-tailed Wilcoxon–Mann–Whitney tests ( g and h ), two-tailed t -distribution with n − 2 d.f. ( i and j ). n = 3 biological replicates ( d – f and k ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Methylation, Control, Binding Assay, Two Tailed Test, MANN-WHITNEY, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Sequence alignment of human MBD family proteins (MBD1-6 and MeCP2) showing differences in the MBD region. b , Validation of Flag-MBD5 or Flag-MBD6 overexpressing mESCs using RT-qPCR. Averaged transcript expressions were indicated. Comparisons against “WT” group. c , Representative gel image quantifying nucleic acids crosslinked to MBD5 or MBD6. Comparisons against “RNase-, DNase-” group. d , EMSA showing the binding preferences of the purified MBD domain of MBD6 fused to maltose binding protein (MBP) with C, m 5 C, or hm 5 C-containing oligonucleotide probes. The purity of the single-stranded probe (ss probe) and double-stranded probe (ds probe) was validated by electrophoresis. Gradient for MBP-MBD protein was 0, 0.1, 0.5, 2.5, 12, 50, and 100 μM. e , EMSA showing that ASXL1 binding to MBD6 does not affect MBD6-m 5 C binding. MBD6 MBD domain (amino acid 1–100 fused by MBP-His) and ASXL1 ASXH domain (amino acid 1509–1541 fused by His-SUMO) were used. f , m 5 C abundance in MBD5 and MBD6 enriched RNA. g , Overlap between the RNA binding sites of MBD5 and MBD6. h - j , Repeat RNA expression ( h ), H2AK119ub ( i ) or caRNA m 5 C ( j ) level upon Mbd5 or Mbd6 KD. Comparisons against “siNC” group. k , IAP RNA lifetime upon Tet2 KO or Mbd6 KD in mESCs. l , qPCR results assessing Mbd6 or Nsun2 RNA abundance in mESCs. Comparisons against “siNC” groups. m , Chromatin openness measured by DNase-TUNEL assay upon Nsun2 or Mbd6 KD. n - r , Spike-in calibrated ATAC-seq signals in WT (WT [siNC]) and Tet2 KO ( Tet2 KO [siNC]) mESCs, as well as Mbd6 KD ( Tet2 KO siMbd6) and Nsun2 KD ( Tet2 KO siNsun2) in Tet2 -KO mESC cells. Metagene profile at genes regulated by different RNA polymerases ( n ), protein-coding genes ( o ), overall signal ( p , q ), or clustered ATAC peaks ( r ) were shown. s , Correlation fold changes in spike-in calibrated ATAC-seq signals in Tet2 KO versus WT mESCs, and comparing Nsun2 KD or Mbd6 KD with control Tet2 KO mESCs. t , Venn diagram displaying the significantly up-regulated ATAC-seq peaks in Tet2 KO compared with WT mESC cells, and down-regulated ATAC-seq peaks in Nsun2 KD or Mbd6 KD compared with control mESC cells following Tet2 KO. Bar ( b , c , f , h - j , l , p ) or line centre ( k ) represents mean and whiskers s.d. P values, two-tailed unpaired t test with Welch’s correction ( b , c , f , h - l ), two-tailed t-distribution with n-2 degrees of freedom ( s ). NS, P > 0.05. n = 3 biological replicates ( b - f , h - r ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Sequencing, Biomarker Discovery, Quantitative RT-PCR, Binding Assay, Purification, Electrophoresis, RNA Binding Assay, RNA Expression, TUNEL Assay, Control, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Metagene profile of the spike-in calibrated ATAC-seq signals of WT and Tet2 −/− HSPCs. b , qPCR results validating efficiency of shRNA-enabled Mbd6 KD in HSPCs. c , Colony formation of serial replating assays of HSPCs upon Mbd6 KD. d , Representative flow cytometry results characterizing HSPC frequency in suspension cultures (top, KIT and Lineage as markers, day 7) and differentiation (bottom, CD11b as marker, day14) upon Mbd6 KD. e , f , Spike-in calibrated ATAC-seq signals in WT (shNC WT) and Tet2 -KO (shNC Tet2 KO) HSPCs, as well as in Mbd6 -KD (shMbd6 Tet2 KO) Tet2 -KO HSPCs. Overall levels ( e ) and metagene profiles ( f ) were shown. g , Volcano plot comparing spike-in calibrated ATAC-seq peak signals in Mbd6 -KD versus control Tet2 -KO HSPCs. h , i Spike-in calibrated H2AK119ub levels in WT (shNC WT) and Tet2 -KO (shNC Tet2 KO) HSPCs, as well as in Mbd6 -KD (shMbd6 Tet2 KO) Tet2 -KO HSPCs. Overall levels ( h ) and metagene profiles ( i ) were shown. j , Volcano plot comparing spike-in calibrated H2AK119ub peak signals in Mbd6 KD compared with control Tet2 -KO HSPCs. k , Quantifications of in vitro differentiation and maintenance of HSPCs with Nsun2 , Nsun5 or Trdmt1 KD. l , qPCR results assessing efficacies of shRNA-enabled Nsun2 , Nsun5 or Trdmt1 KD in HSPCs. m , Half life measurements of IAP RNA in HSPCs of different genotypes. WT denotes wild-type, Tet2 KO denotes Tet2 KO, EE mutant lost hm 5 C oxidation activities and HxD mutant is catalytically inactive. Bar ( b , e , h , l , m ) or line centre ( k , m ) represents mean and whiskers s.d. ( b , k - m ) or s.e.m. ( e , h ). P values, Wald test ( g , j ), two-tailed unpaired t test with Welch’s correction ( b , e , h , k - m ). NS, P > 0.05. n = 3 biological replicates ( a - c , e - m ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: shRNA, Flow Cytometry, Suspension, Marker, Control, In Vitro, Mutagenesis, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Schematics of the mixed chimera transplantation assay. b , Quantification of CD45.2 + cells in the PB of recipient mice at different timepoints after transplantation. c , Quantification of CD45.2 + cells in the BM (left) or spleen (right) of recipient mice. d , e , Representative image ( d ) and quantification ( e ) of spleen size isolated from recipient mice at 24 weeks after transplantation. f , g , Colony formation analysis of the serial replating assay ( f ) and flow cytometry analyses of suspension cultures ( g ) of WT and Tet2 −/− HSPCs with (shMbd6) or without (shNC) Mbd6 KD. h , The correlation between changes in ATAC–seq signal and H2AK119ub signal in response to Mbd6 KD in Tet2 -KO HSPCs. i , Integrative Genome Viewer visualization of the H2AK119ub and ATAC signal around the Socs3 or Nfkbia genes in WT, Tet2 KO and shMbd6 after Tet2 KO in HSPCs. The values in parentheses represent the scale of signal in each track. Data are mean ± s.d. ( b , c and e – g ). P values were calculated using two-tailed unpaired t -tests with Welch’s correction ( b , c and e – g ) and two-tailed t -distribution with n − 2 d.f. ( h ). n = 3 biological replicates ( b – g ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Transplantation Assay, Isolation, Flow Cytometry, Suspension, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Proliferation of WT or TET2 -KO K-562 or THP-1 cells with (shMBD6) or without (shNC) MBD6 KD. n = 4. b , The K-562 H2AK119ub level changes after MBD6 KD. n = 3. c , NSG mice were transplanted with K-562 (left) or THP-1 (right) cells and their overall survival is shown as the Kaplan–Meier estimator. n = 5 mice. d , Heat map illustrating the spike-in-calibrated ATAC–seq signals on ATAC–seq peak regions in WT (siNC WT) and TET2 -KO (siNC TET2 KO) K-562 cells, as well as in NSUN2 KD (siNSUN2 TET2 KO) and MBD6 -KD (siMBD6 TET2 KO) TET2 -KO K-562 cells. n = 3. Data are row-normalized using z scores. e , The correlation of changes in ATAC signals between TET2- KO versus WT K-562 cells, and comparing NSUN2 KD or MBD6 KD with control in TET2- KO K-562 cells. f , m 5 C methylation level changes of K-562 cells in different repeat RNA families after TET2 KO. The size of the circle represents the number of loci methylated by m 5 C. g , The H2AK119ub signals around HERVH-int genomic loci. The colour bar shows H2AK119ub signal. Data are mean ± s.d. P values were calculated using two-tailed unpaired t -tests with Welch’s correction ( a and b ), log-rank Mentel–Cox tests ( c ) and two-tailed t -distribution with n − 2 d.f. ( e ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Control, Methylation, Two Tailed Test, Genome Wide

Journal: Nature

Article Title: RNA m 5 C oxidation by TET2 regulates chromatin state and leukaemogenesis

doi: 10.1038/s41586-024-07969-x

Figure Lengend Snippet: a , Representative subcellular fractionation of K-562 cells. Cyt: cytosolic; Nuc: nuclear soluble; Chr: chromatin. b , Overall caRNA and whole-cell RNA abundance relative to spike-in in WT, TET2 -KO and TET2 -KO + MBD6 -KD K-562 cells. c , Metagene profile of the calibrated ATAC-seq signals of WT (siNC WT) and Tet2 KO (siNC Tet2 KO) K-562 cells, as well as in Mbd6 KD (siMbd6 Tet2 KO) Tet2 KO K-562 cells. d , Volcano plot comparing spike-in calibrated ATAC-seq peaks in Mbd6 -KD versus control in Tet2 -KO K-562 cells. e , Metagene profile of the spike-in calibrated H2AK119ub signals of WT (siNC WT) and Tet2 -KO (siNC Tet2 KO) K-562 cells, as well as in Mbd6 -KD (siMbd6 Tet2-KO) Tet2 -KO K-562 cells. f , Volcano plot comparing spike-in calibrated H2AK119ub peaks in Mbd6 -KD compared with control Tet2 -KO K-562 cells. g , Correlation between changes in ATAC and H2AK119ub signals, comparing Mbd6 -KD with control in Tet2 -KO K-562 cells. h , Venn diagram displaying the significantly up-regulated ATAC-seq peaks in Tet2 -KO compared with WT K-562 cells, and down-regulated ATAC-seq peaks in Nsun2 -KD or Mbd6 -KD compared with control Tet2 -KO K-562 cells. i , Overlap of differentially expressed genes upon TET2 KO in control K-562 cells, and upon MBD6 KD in TET2 -KO K-562 cells. Barplot depicting the odds ratio of upDEGs following TET2 KO, in comparison to both upDEGs and downDEGs upon MBD6 KD in TET2 KO K-562 cells. j , KEGG pathway enrichment analysis performed on genes that were upregulated in TET2 -KO in control K-562 cells, and downregulated in MBD6 -KD in TET2 -KO K-562 cells. Larger circles indicate greater significance. k , Correlation of gene expression changes between TET2 KO versus WT, and MBD6 KD versus control in TET2 KO K-562 cells. The genes are associated with various signalling pathways, including Th1 and Th2 cell differentiation, NF-kappa B signalling pathway, and C-type lectin receptor signalling pathways. Error band represents standard errors. Bar represents mean ( b ) and whiskers s.e.m. P values, Wald test ( d , f ), two-tailed Fisher’s Exact Test ( i ), one-sided Fisher’s Exact test ( j ), two-tailed t-distribution with n-2 degrees of freedom ( g ). n = 3 biological replicates ( a - c , e ). The depicted genome-wide data represent an integration of three biological replicates.

Article Snippet: The antibodies used in this study are summarized below: rabbit monoclonal anti-H2AK119ub antibody (Cell Signaling Technology, 8240S, 1:1,000 for western blot, 1:50 for CUT&Tag); rabbit monoclonal anti-H3 antibody (Cell Signaling Technology, 4499S, 1:1,000); mouse monoclonal anti-TET2 antibody (MilliporeSigma, MABE462, 1:500); rabbit monoclonal anti-GAPDH antibody, HRP conjugate (Cell Signaling Technology, 8884S, 1:1,000); rabbit monoclonal anti-DDDDK tag antibody (Abcam, ab205606, 1:1,000 for western blot, 1:50 for immunoprecipitation); rabbit polyclonal anti-SNRP70/U1-70K antibody (Abcam, ab83306, 1:1,000); mouse monoclonal anti-5-methylcytosine antibody (Diagenode, C15200081-100, 1:1,000 for dot blot, 1:50 for meRIP); mouse monoclonal anti-hm 5 C antibody (Diagenode, C15200200-100, clone Mab-31HMC, 1:1,000 for dot blot); rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling Technology, 9733S, only for CUT&Tag experiments, 1:50); mouse monoclonal anti-BAP1 antibody (Santa Cruz, sc-28383, 1:50 for CUT&Tag).

Techniques: Fractionation, Control, Comparison, Gene Expression, Cell Differentiation, Two Tailed Test, Genome Wide

Journal: Developmental cell

Article Title: Developmentally programmed tankyrase activity upregulates β-catenin and licenses progression of embryonic genome activation

doi: 10.1016/j.devcel.2020.04.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PTEN (D4.3) XP Rabbit mAb antibody , Cell Signaling Technology , 9188; RRID:AB_2253290.

Techniques: Recombinant, Electron Microscopy, Imaging, Isolation, SYBR Green Assay, Sequencing, Mutagenesis, DNA Library Preparation, Purification, Negative Control, Control, Software

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: (A) The experimental approach. (B) GATA3, LHX1, WT1, and HNF4A expression in RCC cells treated with Dec to deplete DNMT1 or UNC1215 to inhibit L3MBTL3. Dec 0.5 μM or UNC1215 1.0 μM added days 1, 2, and 3 each week to RCC cells ACHN and SKRC45. Gene expression by qRT-PCR relative to vehicle-treated control. Plotted are means ± SD for three independent experiments, *p < 0.01, two-sided t test drug versus vehicle. (C) Western blot for MYC (master regulator of proliferation) and p27/CDKN1B (mediates cell-cycle exits by differentiation). Lanes were run on the same gel but were non-contiguous as indicated. (D) Giemsa-stained cytospin preparations of cells harvested on day 5. Scale bar (white line), 12.5 μM; magnification, 400×. (E) Cell counts by automated counter. Plotted are means ± SD from three independent experiments.

Article Snippet: Western analyses of PVDF membranes utilized established protocols and antibodies for DNMT1 (Abcam #Ab54759), mouse anti-flag (Sigma cat# F3165-.2MG), rabbit anti-PBRM1 (ABCAM cat# ab86156), rabbit anti c-MYC (Cell signaling cat# 5605), rabbit anti-p27/CDKN1B (Cell signaling Ab cat# 3833) and β-actin (Sigma, #a3854).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control, Western Blot, Staining

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Western analyses of PVDF membranes utilized established protocols and antibodies for DNMT1 (Abcam #Ab54759), mouse anti-flag (Sigma cat# F3165-.2MG), rabbit anti-PBRM1 (ABCAM cat# ab86156), rabbit anti c-MYC (Cell signaling cat# 5605), rabbit anti-p27/CDKN1B (Cell signaling Ab cat# 3833) and β-actin (Sigma, #a3854).

Techniques: Virus, Recombinant, Protease Inhibitor, cDNA Synthesis, DNA Purification, Software

Journal: Developmental cell

Article Title: Developmentally programmed tankyrase activity upregulates β-catenin and licenses progression of embryonic genome activation

doi: 10.1016/j.devcel.2020.04.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-eIF2α , Cell Signaling , 5324; RRID:AB_10692650.

Techniques: Recombinant, Electron Microscopy, Imaging, Isolation, SYBR Green Assay, Sequencing, Mutagenesis, DNA Library Preparation, Purification, Negative Control, Control, Software

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: Migratory DCs associate with tumor immune status, patient prognosis, and immunotherapeutic response in patients with cancer (A and B) Representative images of multiplex immunostaining of migratory DC and T cell markers on tumor sections derived from immune-“hot” and “cold” patients with PDAC/NSCLC are given. (C and D) Correlation analysis between migratory DC population and overall survival across our collected PDAC and NSCLC patient cohorts ( n = 66 patients with PDAC, n = 65 patients with NSCLC). (E and F) ROC curves represent the accuracy of migratory DCs and CD3 + CD8 + T cells in predicting overall survival in patients with PDAC and NSCLC. (G and H) Spatial analysis of multiplex IF images from each group. Magnified images are given. (I and J) Stack bar charts show the density of CD3 + CD8 + T cells at varying distances from migratory DCs within both hot and cold tumors from patients with PDAC/NSCLC. (K) Representative images of multiplex immunostaining of migratory DC and T cell markers on tumor sections from NSCLC immunotherapy non-responder and responder. (L) High population of migratory DCs correlated with better progression-free survival in patients with advanced NSCLC treated with anti-PD-1 ( n = 19 patients). (M) ROC curves illustrate the predictive accuracy of migratory DCs and CD3 + CD8 + T cells for progression-free survival in patients with advanced NSCLC treated with anti-PD-1. (N) Spatial proximity analysis of multiplex IF images from each group. Magnified image is given. (O) Stack bar chart illustrates the density of CD3 + CD8 + T cells at different distances from migratory DCs in each group. (C, D, and L) Log rank (Mantel-Cox). Scale bars in (A), (B), and (K) represent 50 μm. See also Figure S1 and Tables S1 , , and .

Article Snippet: rabbit mAb against mouse CD8 , CST , Cat# 98941S; RRID:AB_2756376.

Techniques: Multiplex Assay, Immunostaining, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: cNP cancer cell @MV DC treatment triggers DC maturation and CD8 + T and NK cell activation in vitro (A and B) FACS analysis measured CD86 expression in immature human/mouse DCs after treatments. Bar charts display the percentage of CD11c + CD86 + DCs per group ( n = 3 independent experiments). (C and D) FACS analysis examined human/murine CD3 + CD8 + T cell proliferation after treatments ( n = 3 independent experiments). (E–H) FACS analysis assessed human/murine CD3 + CD8 + T cell activation after treatments. Bar charts show the percentage of CD3 + CD8 + CD69 + T cells or CD3 + CD8 + IFN-γ + T cells per group ( n = 3 independent experiments). (I–L) FACS analysis assessed human/murine NK cell activation after treatments. Graphs show the percentage of CD3 − CD56 + CD69 + , CD3 − NK1.1 + CD69 + , CD3 − CD56 + IFN-γ + , or CD3 − NK1.1 + IFN-γ + relative to the total human/murine NK population in each group ( n = 3 independent experiments). (M and N) Schematic diagrams illustrate LDH release assays conducted on PDAC patient-derived tumor organoids (T, target), using varying ratios of CD3 + CD8 + T cells (E, effector) pre-treated with the indicated treatments (M), or co-cultured with hDCs pre-treated with the indicated treatments (N). Bar charts indicate the killing efficiency percentage at each E:T ratio among different groups ( n = 3 independent experiments). (O and P) Cytotoxicity LDH release assays on DT6066 cells were conducted using different ratios of CD3 + CD8 + T cells that had been either pre-treated with the indicated treatments (O) or co-cultured with DCs pre-treated with the indicated treatments (P) ( n = 3 independent experiments). (A–L) One-way ANOVA. (M–P) Two-way ANOVA. See also Figure S3 .

Article Snippet: rabbit mAb against mouse CD8 , CST , Cat# 98941S; RRID:AB_2756376.

Techniques: Activation Assay, In Vitro, Expressing, Derivative Assay, Cell Culture

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet: cNP DT6066 @MV DC treatment converts immune-cold pancreatic tumors into hot tumors, reduces hypoxia, and enhances blood vessel function (A) FACS analysis of CD11c + CD86 + DCs in orthotopic DT6066 pancreatic tumors after treatments, with a bar chart showing their percentage relative to total lymphocytes ( n = 3 mice per group). (B) FACS analysis of CD3 + CD4 + CD69 + T cells in orthotopic pancreatic tumors after treatments, with a bar chart showing their percentage relative to total CD3 + CD4 + T cells in each group ( n = 3 mice per group). (C) FACS analysis of CD3 + CD8 + CD69 + T cells in orthotopic pancreatic tumors after treatments, with a bar chart showing their percentage relative to total CD3 + CD8 + T cells in each group ( n = 3 mice per group). (D) Representative images of CD11c and CD86 co-immunostaining in tumor sections from each treatment group. Bar chart shows the quantification of CD11c + CD86 + DC × 10 3 per cm 2 in each group ( n = 3 mice per group). (E and F) Representative images of co-immunostaining for CD3 and CD4 (E) or CD8 (F) in tumor sections from each treatment group ( n = 3 mice per group). (G) FACS analysis of CD3 − NK1.1 + NK cells or CD3 − NK1.1 + CD69 + NK cells in orthotopic pancreatic tumors after treatments. Bar charts show their percentage relative to total CD3 − cells (left) or CD3 − NK1.1 + NK cells (right) in each group ( n = 3 mice per group). (H–J) Cytotoxicity LDH release assays of DT6066 cells after co-culture with various ratios of CD3 + CD8 + T cells isolated from tumors (H), lymph nodes (I), or spleens (J) of orthotopic DT6066 pancreatic tumor-bearing mice after treatments ( n = 3 mice per group). (K) FACS analysis of migratory DCs in orthotopic pancreatic tumors and TDLNs from mice treated as indicated. Bar charts show CD86 + CD103 + DCs as a percentage of CD11c + DCs in each group ( n = 3 mice per group). (L) Representative images of CD11c/CD86/CD103 triple immunostaining on paired tumor and TDLN sections in each group. Bar chart shows the percentage of CD11c + CD86 + CD103 + migratory DC × 10 3 per cm 2 in tumors (left) or TDLNs (right) from each group ( n = 3 mice per group). (M–O) Representative microbubble contrast ultrasound images are shown, and bar charts show quantification across the entire tumors, including tumor cores ( n = 3 mice per group). (P) Representative IHC staining of endomucin in tumor sections from each treatment group. Bar chart shows blood vessel diameter (left) or number of blood vessels (right) per cm 2 in each group ( n = 3 mice per group). (Q) Representative image of GLUT1 and endomucin co-immunostaining in tumor sections from each group. Bar chart shows the relative GLUT1 intensity per group ( n = 3 mice per group). (A–G, K, L, and N–Q) One-way ANOVA. (H–J) Two-way ANOVA. Scale bars in (D), (E), (F), and (L) represent 50 μm. (M) 1 cm. (P and Q) 100 μm. See also Figures S5 and .

Article Snippet: rabbit mAb against mouse CD8 , CST , Cat# 98941S; RRID:AB_2756376.

Techniques: Immunostaining, Co-Culture Assay, Isolation, Triple Immunostaining, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: Mitochondrial DNA-boosted dendritic cell-based nanovaccination triggers antitumor immunity in lung and pancreatic cancers

doi: 10.1016/j.xcrm.2024.101648

Figure Lengend Snippet:

Article Snippet: rabbit mAb against mouse CD8 , CST , Cat# 98941S; RRID:AB_2756376.

Techniques: Virus, Recombinant, Negative Staining, Lysis, Control, Phospho-proteomics, Cell Isolation, DNA Purification, DNA Extraction, Bicinchoninic Acid Protein Assay, Protein Purification, Magnetic Beads, Software

Journal: Cell reports

Article Title: A multiplexed in vivo approach to identify driver genes in small cell lung cancer

doi: 10.1016/j.celrep.2023.111990

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-GFP (D5.1) Monoclonal , Cell Signaling Technology , Cat#2956; RRID:AB_1196615.

Techniques: Plasmid Preparation, Virus, Recombinant, Modification, Labeling, Amplification, Polymer, Staining, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Simple Western, Membrane, DNA Extraction, Purification, Sequencing, Generated, Software