Journal: iScience

Article Title: Molecular and cellular basis of hyperassembly and protein aggregation driven by a rare pathogenic mutation in DDX3X

doi: 10.1016/j.isci.2021.102841

Figure Lengend Snippet:

Article Snippet: Plasmid: pEGFP-C1 , Addgene , 6084-1.

Techniques: Virus, cDNA Library Assay, Recombinant, Proliferation Assay, SYBR Green Assay, Mutagenesis, In Situ, Control, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Synthetic multicolor antigen-stabilizable nanobody platform for intersectional labelling and functional imaging

doi: 10.1101/2025.10.27.684934

Figure Lengend Snippet: (a) Change in fluorescence intensity of the cell co-expressing jRGECO1a-Fb LAG30 (red) and mEGFP (green) in response to 5 μM ionomycin. (b) Change in fluorescence intensity of the cell co-expressing jRGECO1a-Fb LAG30 (red) and Green Pegassos pyruvate biosensor (green) in response to 1 mM pyruvate. (c) Change in fluorescence intensity of the cell co-expressing jRGECO1a-Fb LAG30 (red) and PyronicSF pyruvate biosensor (green) in response to 10 mM pyruvate. (d) Change in fluorescence intensity of the cell co-expressing jRGECO1a-Fb LAG30 (red) and iGlucoSnFr biosensor (green) in response to 20 mM pyruvate. (a-d) Scale bar, 40 μm. (e) Contrast of mEGFP co-expressed with jRGECO1a-Fb LAG30 ( n=10 ) after addition of 5 μM ionomycin. (f) Contrast of Green Pegassos only ( n=9 ) and Green Pegassos co-expressed with jRGECO1a-Fb LAG30 ( n=9 ) after addition of 1 mM pyruvate. (g) Contrast of PyronicSF only ( n=10 ) and PyronicSF co-expressed with jRGECO1a-Fb LAG30 ( n=9 ) after the addition of 10 mM pyruvate. (h) Contrast of iGlucoSnFr only ( n=11 ) and iGlucoSnFr co-expressed with jRGECO1a-Fb LAG30 ( n=13 ) after the addition of 20 mM glucose. (i) Contrast of jRGECO1a ( n=16 ) and jRGECO1a-Fb LAG30 only ( n=10 ) or jRGECO1a-Fb LAG30 co-expressed with Green Pegassos ( n=9 ), PyronicSF ( n=9 ) or iGlucoSnFr ( n=13 ). In (b-d), the following filters were used: for imaging mEGFP, Green Pegassos, PyronicSF, and iGlucoSnFr 480/40 nm excitation and 535/40 nm emission; for imaging jRGECO1a-Fb LAG30 575/25 nm excitation and 615/30 nm emission.

Article Snippet: Anti-mCherry Nb was amplified from the pGEX6P1-mCherry-Nanobody (LaM-2) (Addgene no.162276), mClover3 was amplified from pNCS-mClover3 (Addgene no.74236). jRGECO1a biosensor was amplified from a pGP-CMV-NES-jRGECO1a (Addgene no.61563) plasmid and inserted into the NIR-Fb LAG30 (Addgene no.220740) plasmid by NEBuilder HiFi DNA Assembly Kit instead of miRFP670nano3.

Techniques: Fluorescence, Expressing, Imaging

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Schematic of HOTTIP ChIRP-LC-MS/MS/WB workflow. (B) Left: one-step blue protein gel staining of proteins isolated from MOLM13 cells by ChIRP using HOTTIP or control LacZ titling probes. Right: partial list of unique polypeptides identified in the HOTTIP , but not in the LacZ ChIRP-LC-MS/MS. (C) Overrepresentation analysis of enriched HOTTIP -interacting protein classes using the DAVID database. The ratio of each protein class represented in the HOTTIP -associated proteome was calculated and statistical significance was ranked according to the Benjamini-Hochberg-corrected p value (p ≤ 0.05). (D) ChIRP-WB validation of HOTTIP -associated proteins. (E) Biotin pull-down of CTCF mutants by biotinylated HOTTIP .

Article Snippet: pTre-Tight-luciferase- HOTTIP , Addgene , Addgene Plasmid #65490; RRID:Addgene_65490.

Techniques: Liquid Chromatography with Mass Spectroscopy, Staining, Isolation, Control, Biomarker Discovery

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Heatmap of CTCF binding globally (top) and at HOTTIP /CTCF co-bound promoters (middle) and intergenic regions (bottom) in WT and HOTTIP −/−; MOLM13 cells. p value calculated by Kolmogorov-Smirnov (K-S) test. (B) Top enriched TF-binding motifs in HOTTIP /CTCF co-occupied peaks according to de novo motif analysis. (C) Histogram of the distribution of HOTTIP and TF motifs within HOTTIP /CTCF co-occupied peaks. (D) Heatmap of R-loop peaks identified by DRIP-seq globally (left), at HOTTIP -bound regions (middle) and at HOTTIP /CTCF co-bound regions (right) in WT and HOTTIP −/−; MOLM13 cells. p value calculated by K-S test. (E) Overlap of HOTTIP -binding peaks identified by ChIRP-seq and CTCF/cohesin co-occupied sites (left) or cohesin independent CTCF sites (right) identified by CTCF and RAD21 ChIP-seq in MOLM13 cells. (F) Overlap of total reduced HOTTIP peaks and all decreased CTCF/cohesin co-bound sites in the genome comparing WT and HOTTIP −/−; MOLM13 cells. (G) Heatmap of RAD21, SA1, and SA2 binding identified by ChIP-seq at HOTTIP /CTCF co-bound sites in WT and HOTTIP −/−; MOLM13 cells. p value calculated by K-S test. (H) Heatmap of H3K27me3 and H3K4me3 profiles identified by ChIP-seq at HOTTIP /CTCF co-bound sites in WT and HOTTIP −/−; MOLM13 cells.

Article Snippet: pTre-Tight-luciferase- HOTTIP , Addgene , Addgene Plasmid #65490; RRID:Addgene_65490.

Techniques: Binding Assay, ChIP-sequencing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Heatmap of ≥2-fold downregulated genes in MOLM13 cells upon HOTTIP −/−; as determined by RNA-seq. (B) GSEA of downregulated genes after HOTTIP −/−; . (C) Overlap of TADs identified by Hi-C in WT and HOTTIP −/−; MOLM13 cells. The domain score of an altered TAD was normalized (quantile-normalization) by subtracting the mean of all TAD Hi-C signals. ANOVA was used to identify significantly altered TADs (Bonferroni-corrected p value < 0.05). (D) GO analysis of genes encompassed by the decreased TADs upon HOTTIP −/−; . (E) Hi-C interaction map at the CTNNB1 locus comparing WT and HOTTIP −/−; MOLM13 cells. (F) ATAC-seq analysis of CTNNB1 in WT and HOTTIP −/−; MOLM13 cells. (G and H) NG Capture-C analysis of CTNNB1 (G) or MYC (H) promoter interactions, CTCF ChIP-seq and HOTTIP ChIRP-seq in WT and HOTTIP −/−; MOLM13 cells. Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively.

Article Snippet: pTre-Tight-luciferase- HOTTIP , Addgene , Addgene Plasmid #65490; RRID:Addgene_65490.

Techniques: RNA Sequencing, Hi-C, Capture-C, ChIP-sequencing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Heatmap of ≥2-fold upregulated genes in BM LSK cells from Hottip -Tg mice. (B) GSEA of upregulated genes in LSK cells upon Hottip activation. (C) Heatmap of CTCF binding, from ChIP-seq, and Hottip binding, from ChIRP-seq, at CTCF/ Hottip co-occupied sites in BM LK cells from WT and Hottip -Tg mice. (D) CTCF ChIP-seq and Hottip ChIRP-seq binding profiles at the Ctnnb1 locus in WT and Hottip -Tg LK cells. (E) Hi-C interaction maps at the Ctnnb1 locus in WT and Hottip -Tg BM LK cells. CTCF-bound TAD boundaries indicated by red arrows. (F) Overlap of WT and Hottip -Tg Hi-C signals from (E). (G) ATAC-seq analysis of Ctnnb1 in WT and Hottip -Tg BM LSKs. (H) tSNE visualization of BM LK cell subsets from Hottip -Tg (red) and WT (blue) mice by scRNA-seq. LT-HSC, ST-HSC, and MPP populations encompassed by blue circle. (I) Trajectory inference branches/clusters were generated based on the expression levels of lineage-associated genes in cell clusters (left) from WT and Hottip -Tg BM LK cells. Sub-population cell density analysis (right) correlated with the enriched cell number of each population. Higher cell densities shown in dark red. (J) The levels of Myc and Hoxa9 in each cell subset along HSC to MEP differentiation in WT and Hottip -Tg BM LK cells by scRNA-seq. The FDR-corrected p value ≤0.05 by binomial and hypergeometric test. (K) Relative cell numbers in each cell subset along HSC to MEP differentiation in WT and Hottip -Tg BM LK cells by scRNA-seq. The FDR-corrected p value ≤0.05 by binomial and hypergeometric test. (L) GO analysis of upregulated genes in LT- and ST-HSC populations upon Hottip activation by scRNA-seq.

Article Snippet: pTre-Tight-luciferase- HOTTIP , Addgene , Addgene Plasmid #65490; RRID:Addgene_65490.

Techniques: Activation Assay, Binding Assay, ChIP-sequencing, Hi-C, Generated, Expressing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) CTCF ChIP-seq and HOTTIP ChIRP-seq-binding profiles at the upstream CBS s of CTNNB1 in WT and HOTTIP −/−; MOLM13 cells. (B) Schematic of probes used in EMSA. The G-4 sequence in HOTTIP , the C-rich HOTTIP -binding motif in the CBS-u2 sequence, and the CTCF-binding motif are shown in red, green, and yellow highlighting, respectively. (C) EMSA of a Cy5-labeled HOTTIP RNA probe (red) and a Cy3-labeled CTNNB1 CBS-u2 probe (green). Orange indicates R-loop formation. RNase A, RNase H, DNase I, and S9.6 antibody added as indicated. (D) Schematic of RIDP procedure (left). RIDP RT-qPCR of HOTTIP precipitated by probes targeting the template or non-template strand of the CBS-u2 site (in relation to the direction of CTNNB1 transcription) or targeting the negative control CBS at ACTB in WT, HOTTIP −/−; or RNase H treatment (right). Data presented as mean ± SD; ***p ≤ 0.001. (E) CTCF and cohesin-binding ChIP-seq, R-loop DRIP-seq, HOTTIP -binding ChIRP-seq and nascent RNA GRO-seq profiles at the CTNNB1 locus in WT and HOTTIP −/−; MOLM13 cells. (F) H3K4me3 and H3K27me3 ChIP-seq enrichment profiles at the CTNNB1 locus in WT and HOTTIP −/−; MOLM13 cells.

Article Snippet: pTre-Tight-luciferase- HOTTIP , Addgene , Addgene Plasmid #65490; RRID:Addgene_65490.

Techniques: ChIP-sequencing, Binding Assay, Sequencing, Labeling, Quantitative RT-PCR, Negative Control

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Schematic of the CTNNB1 locus showing the locations of CBS s, sub-TADs, and TAD. (B) CTCF ChIP-qPCR analysis of the indicated sites in WT, CBS-u1 −/−; and CBS-u2 −/−; MOLM13 cells. (C) RT-qPCR analysis of the indicated transcripts in WT, CBS-u1 −/−; , and CBS-u2 −/−; MOLM13 cells. (D) Proliferation of WT, CBS-u1 −/−; , and CBS-u2 −/−; MOLM13 cells. (E) HOTTIP ChIRP-qPCR analysis of the indicated sites in WT, CBS-u1 −/−; , and CBS-u2 −/−; MOLM13 cells. (F) DRIP-qPCR analysis of the indicated sites in WT, CBS-u1 −/−; , and CBS-u2 −/−; MOLM13 cells. (G) NG Capture-C analysis of CTNNB1 promoter interactions in WT and CBS-u2 −/−; MOLM13 cells. Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively. Capture-C data were aligned with CTCF, H3K4me3, and H3K27me3 ChIP-seq profiles in the CTNNB1 locus in WT, HOTTIP −/−; , and CBS-u2 −/−; MOLM13 cells. (H) Kaplan-Meier survival curves of NSG mice transplanted with WT, CTNNB1 +/− , and CBS-u2 −/−; MOLM13 cells. n = 5. (I) hCD45 + cells chimerism in the BM and PB of NSG mice transplanted with WT, CTNNB1 +/− , and CBS-u2 −/−; MOLM13 cells. n = 3. (J) Kaplan-Meier survival curves of NSG mice transplanted with WT or CBS-u2 −/−; primary AML cells carrying MLL + (LPP4) or NPM1 C+ FLT3-ITD + (#974) mutations. n = 4. (K) hCD45 + cell chimerism in the BM of NSG mice transplanted with WT or CBS-u2 −/−; primary AML cells. Data in (B)–(F) and (H)–(K) are presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Article Snippet: pTre-Tight-luciferase- HOTTIP , Addgene , Addgene Plasmid #65490; RRID:Addgene_65490.

Techniques: ChIP-qPCR, Quantitative RT-PCR, Capture-C, ChIP-sequencing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: (A) Schematic of the CTNNB1 locus. Shown is the CBS-u2 sequence with CTCF (yellow) and HOTTIP (green) motifs and the sgRNA target site (red) indicated. (B) RT-qPCR analysis of β-catenin and its target gene expression upon expression of dCas9-RNase H or dCas9-RNase H D210N with or without CBS-u2 -targeted sgRNA or exogenous β-catenin expression in MOLM13 cells. (C) Proliferation of WT, CBS-u2 RH-WT , CBS-u2 RH-Mut , and β-catenin-rescued CBS-u2 RH-WT MOLM13 cells. (D) CTCF ChIP-qPCR analysis of the indicated sites in WT, CBS-u2 RH-WT , CBS-u2 RH-Mut , and β-catenin-rescued CBS-u2 RH-WT MOLM13 cells. (E) HOTTIP ChIRP qPCR analysis of the indicated sites in WT, CBS-u2 RH-WT , CBS-u2 RH-Mut , and β-catenin-rescued CBS-u2 RH-WT MOLM13 cells. (F) DRIP-qPCR analysis of the indicated sites in WT, CBS-u2 RH-WT , CBS-u2 RH-Mut , and β-catenin-rescued CBS-u2 RH-WT MOLM13 cells. (G) NG Capture-C analysis of CTNNB1 promoter interactions upon expression of CBS-u2 RH-WT . Solid purple and dashed red lines indicate unchanged and reduced interactions, respectively. Capture-C data were aligned with CTCF, H3K4me3, and H3K27me3 ChIP-seq profiles in the CTNNB1 locus in WT, HOTTIP −/−; , and CBS-u2 RH-WT MOLM13 cells. (H) Kaplan-Meier survival curves of NBSGW mice transplanted with WT, CBS-u2 RH-Mut , or CBS-u2 RH-WT MOLM13 cells. (I) Kaplan-Meier survival curves of NSG mice transplanted with CBS-u2 RH-WT or CBS-u2 RH-Mut OCI-AML3 cells. Mice were sacrificed when they were paralyzed due to the disease. Data in (B)–(F) and (H)–(I) are presented as mean ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Article Snippet: pTre-Tight-luciferase- HOTTIP , Addgene , Addgene Plasmid #65490; RRID:Addgene_65490.

Techniques: Sequencing, Quantitative RT-PCR, Targeted Gene Expression, Expressing, ChIP-qPCR, Capture-C, ChIP-sequencing

Journal: Molecular cell

Article Title: HOTTIP -dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

doi: 10.1016/j.molcel.2022.01.014

Figure Lengend Snippet: Key resources table

Article Snippet: pTre-Tight-luciferase- HOTTIP , Addgene , Addgene Plasmid #65490; RRID:Addgene_65490.

Techniques: Control, Recombinant, CRISPR, Electroporation, Protease Inhibitor, Staining, Transfection, Reverse Transcription, Purification, Plasmid Preparation, DNA Library Preparation, Multiplex Assay, Hybridization, Library Quantification, Sensitive Assay, SYBR Green Assay, Transgenic Assay, Software