human embryonic kidney 293 cells  (ATCC)

Journal: Human Genetics and Genomics Advances

Article Title: Fine mapping regulatory variants by characterizing native CpG methylation with nanopore long-read sequencing

doi: 10.1016/j.xhgg.2025.100532

Figure Lengend Snippet: Adaptive sampling nanopore long-read sequencing targeting GWAS and mQTL loci (A) Proportional Venn diagram of the target SNP with TCGA mQTL signals and GWAS risk significance. (B) Sequencing coverage summarized by chromosomes with AS run in the 22Rv1 genome. (C) Correlation between the target proportion and enrichment fold across the 22 autosomes. (D and E) Distribution of dual methylation calling for the CpG sites in (D) or outside of (E) the CpG island in chromosome 19. The percentages highlight the proportion of highly confident modified sites within the nearby squares. (F) Circular plot of 5hmC modification percentage (green line, outermost circle), DMR methylation difference (red and blue scatters, middle circle), and the DMR density (red and blue peaks, innermost circle).

Article Snippet: The 22Rv1 (RRID: CVCL_1045) cell line was obtained from the ATCC and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).

Techniques: Sampling, Sequencing, Methylation, Modification

Journal: Human Genetics and Genomics Advances

Article Title: Fine mapping regulatory variants by characterizing native CpG methylation with nanopore long-read sequencing

doi: 10.1016/j.xhgg.2025.100532

Figure Lengend Snippet: Haplotype specific methylation regions co-localized with allelic accessible region at GWAS and mQTL loci in 22Rv1 cells (A) Exemplary differential methylated region (DMR) identified near the GWAS risk variant rs7247241 and chromatin accessibility profiling with the rs7247241 alleles in 22Rv1 cells. (B) GTEx eQTL results for rs7247241 genotype and PPP1R14A expression in prostate tissue. (C) Exemplary DMR identified near the mQTL variant rs2113417 and chromatin accessibility profiling with the rs2113417 alleles in 22Rv1 cells. (D) GTEx eQTL results for rs2113417 genotype and KCNIP3 expression in prostate tissue. (E) TCGA mQTL results for rs2113417 in prostate cancer tissue. (F) Correlation between Tn5 insertion coverage and methylation percentage in the chromatin-accessible (ATAC-seq peak) regions in 22Rv1 cells. Boxplots display the distribution of values across groups: the horizontal line indicates the median, the box spans the interquartile range (25th–75th percentile), and whiskers extend to values within 1.5 × IQR. Simple linear regression test was used to compare the gene expression and methylation difference between genotypes.

Article Snippet: The 22Rv1 (RRID: CVCL_1045) cell line was obtained from the ATCC and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).

Techniques: Methylation, Variant Assay, Expressing, Gene Expression

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Talin contains tensin-binding sites in the R3, R4, R7R8, and R11 domain. (A) Schematic representation of talin with the aa numbers of each domain indicated. (B) Schematic representation of tensin3. Talin-binding site (TBS), aa 692–718. (C) Mitochondrial targeting talin1 full-length and deletion constructs. The indicated talin sequences were inserted between EGFP (GFP) and cBAK, with the aa number of talin regions indicated below. (D) Constructs shown in C were expressed with mCh-IDR in NIH3T3 cells. Black boxes indicate colocalization, and gray boxes indicate no association. (E) Western blotting of mitochondrial pulldown experiments. Constructs as used in D were expressed in HEK293T cells. Whole cell lysates (wcl) and purified mitochondria were immunoblotted. Note that the double band for mCh-IDR is due to known mCherry degradation. (F) Representation of additional talin1 constructs. (G–I) NIH3T3 cells expressing the constructs shown in C and F with mCh-IDR. (J) Summary table of the mitochondrial targeting assays. All results are collected from three independent experiments. Scale bars (D and G–I), 5 μm. aa, amino acid. Source data are available for this figure: .

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Binding Assay, Construct, Western Blot, Purification, Expressing

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Tensin3 binds multiple talin rod domains. (A–C) Western blotting of the mitochondrial pulldown experiments in HEK293T cells with the same constructs used in , showing that R3, R4, and R11 interact with mCh-IDR. (D) Representative images of NIH3T3 cells co-expressing mCh-TNS3-cBAK with GFP-vector, GFP-R1R2, GFP-R1R3, GFP-R4R6, GFP-R5R6, GFP-R7R8, GFP-R9R10, GFP-R11DD, or GFP-R12DD, respectively. Black boxes indicate colocalization, and gray boxes indicate no association. Note that TNS3-cBAK recruits R1R3, R4R6, R7R8, and R11DD to the mitochondria, but not GFP-vector, R1R2, R5R6, R9R10, or R12DD. (E) Summary table of the MTS experiments in D. (F–I) Representative images of NIH3T3 cells co-expressing GFP-R1R3-cBAK (F), GFP-R4R6-cBAK (G), GFP-R7R8-cBAK (H), or GFP-R11DD-cBAK (I) with mCh-IDR-WT or mCh-IDR-L702E, respectively. Note that L702E abolishes mCh-IDR interactions with all talin1 truncation constructs. (J) Images of cells co-expressing mCh-TNS3-cBAK (WT or L702E) with GFP-TLN1-E1770A, respectively. L702E abolishes TNS3 colocalization with TLN1-E1770A at the mitochondria. All experiments are performed three times. Scale bars, 5 μm. Source data are available for this figure: .

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Construct, Expressing, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: Talin–tensin3 interactions regulate fibrillar adhesion formation and tensin3 phase separation

doi: 10.1083/jcb.202503155

Figure Lengend Snippet: Tensin3 LLPS is controlled by talin activity in response to rigidity sensing. (A) Confocal images of NIH3T3 cells expressing mCh-TNS3-WT or mCh-TNS3-L702E plated overnight on FN-coated 1.5 or 28 kPa PDMS dishes. (B) Quantification of the mean TNS3 condensate number in A. n = 83 (WT/1.5 kPa), 80 (WT/28 kPa), 83 (L702E/1.5 kPa), and 89 (L702E/28 kPa) cells. Note that condensates that are larger than 0.1 μm 2 with a circularity between 0.7 and 1 were quantified. (C) Images of endogenous tensin3 or GFP-TNS3 (green) with endogenous paxillin (magenta) in HFF cells (top panel) and NIH3T3 cells (bottom panel) plated on FN-coated glass, respectively. The dashed boxes are zoomed to the right, with condensates indicated by yellow arrows. (D) Quantification of the circularity of overexpressed GFP-TNS3 condensates in NIH3T3 cells, endogenous tensin3 condensates in HFF cells, and tensin3 adhesions in HFF cells. The boxes represent the 25–75th percentiles with the median indicated; the whiskers indicate the range of values. n = 717 (overexpressed condensates, 17 cells), 192 (endogenous condensates, 29 cells), and 4,883 (tensin3 adhesions, 33 cells). (E) Representative images of HFF cells plated on FN-coated glass (left panel). The dashed boxes are zoomed to the right panel, with tensin3 condensates (green) indicated by white arrows and a line profile below for the yellow arrow line. The lipid membrane is labeled with WGA (blue); the active β1 integrin (magenta) is stained using 9EG7 antibody. Note that the tensin3 condensate is not colocalized with vesicle structures indicated by WGA and active β1 integrin. (F) Images (background-subtracted) of HFF cells plated overnight on FN-coated 1.5 or 28 kPa PDMS dishes. Endogenous tensin3 (green) and paxillin (magenta) were visualized by staining. The dashed boxes are zoomed in below, with condensates indicated by yellow arrows. (G) Quantification of the mean number of endogenous tensin3 condensates in F. n = 33 (1.5 kPa) and 40 (28 kPa) cells. (H) Mitochondrial pulldown experiments using HEK293T cells expressing GFP-TLN1-cBAK (WT or E1770A) and mCh-IDR. (I) Quantification of H pooled from three replicates. Data are normalized to WT. Error bars are the SEM. All data are collected from three independent experiments. * indicates P < 0.05, and **** indicates P <0.0001 (B and D: Kruskal–Wallis test with Dunn’s multiple comparisons; G: Mann–Whitney test; I: unpaired t test). Scale bar: 10 µm (A, C, and F) and 5 µm (E). Source data are available for this figure: .

Article Snippet: NIH3T3 mouse fibroblasts, HEK293T human epithelial cells, and U2OS human osteosarcoma cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Expressing, Membrane, Labeling, Staining, MANN-WHITNEY