Journal: eLife

Article Title: Human dynein–dynactin is a fast processive motor in living cells

doi: 10.7554/eLife.94963

Figure Lengend Snippet: ( A ) Montage of representative fields of view of the CRISPR-engineered p50-EGFP clone in comparison to the montage of DHC-EGFP fields from . For the purpose of comparison, the two cell lines were visualized with identical imaging parameters on the same day and displayed identically in the figure. ( B ) Scatter plots of background-corrected fluorescence intensities of cytoplasmic pools of DHC-EGFP ( n = 31 cells) and p50-EGFP ( n = 13 cells) from ( A ). ( C ) Montage of fields of view p50-EGFP clonal cells visualized with longer exposure times (500 ms) than in ( A ). Error bars are mean values ± standard deviations. Scale bars, 20 μm. Figure 2—figure supplement 1—source data 1. Excel spreadsheet containing the underlying data and numerical values for plots in .

Article Snippet: Production of EGFP-tagged p50 in the knock-in cell line was confirmed via western blot for p50 and integration of the EGFP repair cassette in the DHC-EGFP knock-in cell line was confirmed by amplicon sequencing (Plasmidsaurus).

Techniques: CRISPR, Comparison, Imaging, Fluorescence

Journal: eLife

Article Title: Human dynein–dynactin is a fast processive motor in living cells

doi: 10.7554/eLife.94963

Figure Lengend Snippet: ( A ) Still frames from a representative spinning disc confocal time-lapse of a p50-EGFP-expressing HeLa cell. A zoomed view of the boxed region is shown with a kymograph of the tip-tracking event highlighted with the yellow arrow. ( B ) Still frames from a representative total internal reflection fluorescence microscopy (TIRFM) time-lapse of a p50-EGFP-expressing HeLa cell showing the tip-tracking population. A zoomed view of the boxed region is shown with a kymograph of the tip-tracking event highlighted by the yellow arrow. ( C ) Still frame from a representative TIRFM time-lapse of a p50-EGFP-expressing HeLa cell treated with SiR-Tubulin. In the merge image, p50 is green and Sir-Tubulin labeled MTs are magenta. ( D ) Representative kymographs of motile p50 puncta spanning the range of measured velocities (Vel) and run lengths (RL). ( E ) Distribution of p50 velocities ( n = 44 puncta). ( F ) Distribution of p50 run lengths ( n = 41 puncta). ( G ) Distribution of p50 run times ( n = 41 puncta). Scale bars, 10 μm ( A–C ); 1 µm (all insets), 5 µm (horizontal); 1 min (vertical) in the kymographs in A and B; and 1 µm (horizontal); 1 s (vertical) in D. Displayed times are min:s. Figure 2—source data 1. Excel spreadsheet containing the underlying data and numerical values for plots in .

Article Snippet: Production of EGFP-tagged p50 in the knock-in cell line was confirmed via western blot for p50 and integration of the EGFP repair cassette in the DHC-EGFP knock-in cell line was confirmed by amplicon sequencing (Plasmidsaurus).

Techniques: Expressing, Fluorescence, Microscopy, Labeling

Journal: eLife

Article Title: Human dynein–dynactin is a fast processive motor in living cells

doi: 10.7554/eLife.94963

Figure Lengend Snippet: Scatter plots of ( A ) velocities (DHC, n = 100; p50, n = 44), ( B ) run lengths (DHC, n = 81; p50, n = 41), and ( C ) run times (DHC, n = 81; p50, n = 41). Distributions of the background-corrected fluorescence intensities of motile puncta of ( D ) kinesin-1-EGFP transiently expressed in HeLa cells, ( E ) DHC-EGFP, and ( F ) p50-EGFP. The dashed line in each histogram denotes the mean value of the kinesin-1-EGFP dataset. ( G ) Scatter plots of the kinesin-1, DHC, and p50 fluorescence intensities (kinesin-1, n = 90 puncta; DHC, n = 84 puncta; p50, n = 74 puncta). ( H ) PCR of genomic DNA from the DHC-EGP clone used in this study using PCR primers flanking the integration site of the repair cassette. The upper band was extracted and subjected to sequencing, the results of which are shown in . ( I ) Western blot for p50 of cell lysates from the parental HeLa cell line and the p50-EGFP clone used in this study. The tagged p50 runs ~30 kDa larger than the untagged p50 and is expressed at ~5- to 6-fold lower levels than the endogenous p50. Error bars are mean values ± standard deviations. The reported p-values were determined by a randomization method: n.s. is not significant (p > 0.05). Figure 3—source data 1. PowerPoint file containing original image of agarose gel for , indicating the relevant PCR fragments. Figure 3—source data 2. Original file of agarose gel image in . Figure 3—source data 3. PowerPoint file containing original membrane and western blots for , indicating the relevant bands and cell line lysates. Figure 3—source data 4. Original files for western blot in . Figure 3—source data 5. Excel spreadsheet containing the underlying processed data and numerical values for plots in .

Article Snippet: Production of EGFP-tagged p50 in the knock-in cell line was confirmed via western blot for p50 and integration of the EGFP repair cassette in the DHC-EGFP knock-in cell line was confirmed by amplicon sequencing (Plasmidsaurus).

Techniques: Fluorescence, Sequencing, Western Blot, Agarose Gel Electrophoresis, Membrane

Journal: eLife

Article Title: Human dynein–dynactin is a fast processive motor in living cells

doi: 10.7554/eLife.94963

Figure Lengend Snippet: Distributions of background-corrected fluorescence of ( A ) kinesin-1-EGFP expressed in Drosophila melanogaster S2 cells, ( B ) DHC-EGFP, and ( C ) p50-EGFP. The dashed line in each histogram denotes the mean value of the kinesin-1-EGFP dataset. ( D ) Box and whisker plots of the kinesin-1, DHC, and p50 fluorescence intensities (kinesin-1, n = 100 puncta; DHC, n = 71 puncta; p50, n = 38 puncta). The reported p-values were determined by a randomization method: n.s. is not significant (p > 0.05). Figure 3—figure supplement 2—source data 1. Excel spreadsheet containing the underlying data and numerical values for plots in .

Article Snippet: Production of EGFP-tagged p50 in the knock-in cell line was confirmed via western blot for p50 and integration of the EGFP repair cassette in the DHC-EGFP knock-in cell line was confirmed by amplicon sequencing (Plasmidsaurus).

Techniques: Fluorescence, Whisker Assay

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) Difficulty to maintain healthy weight persists despite several therapeutic approaches. TPN, total peripheral nutrition. ( B ) Family pedigree for heterozygous 1377delT NFKB1 variant. ( C ) Schematic illustration of NFKB1 , p50, and p105 coding regions as denoted. Location of 1377delT variant as indicated in red. RHD, Rel homology domain; ARD, ankyrin repeat domain; DD, death domain. ( D ) NFKB1 sequence from PBMCs by Sanger sequencing for healthy donor (HD) on left and CA01 on right. 1377delT frameshift denoted in red.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques: Variant Assay, Sequencing

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) Schematic of protocol used to generate monocytes from iPSCs. ( B ) Morphology and DNA sequence in region of 1377delT NFKB1 variant (highlighted in yellow) for each iPSC line used in this study. ( C ) Flow cytometry of CD45/CD14 and CD16/CD163 on iMONOs generated from each iPSC line used in this study. Data representative of more than 12 iMONO differentiations. Genetic background of iPSC line and presence of heterozygous NFKB1 variant as denoted.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques: Sequencing, Variant Assay, Flow Cytometry, Generated

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) Schematic of protocol for iMONO assay. ( B ) Whole cell iMONO protein lysate (20 μg) analyzed for p105/p50 protein level by Western blot. Source of iPSC and present of NFKB1 variant as indicated. ( C ) Densitometry analysis of Western blot bands from 3 blots generated from 3 independent experiments. P value was calculated by 1-way ANOVA with Holm-Šídák test for multiple comparisons. * P < 0.05.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques: Western Blot, Variant Assay, Generated

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) IL-1β, CXCL1, and CXCL2 measured in supernatant of iMONO cultures with and without LPS. ( B ) IL-12, CCL4, and CCL22 measured in supernatant of iMONO cultures with and without LPS. Parental iPSC line (CA01 or HD) and presence of WT or 1377delT NFKB1 for both alleles as noted. Each data point represents an experimental replicate. P value was calculated by 2-way ANOVA with Holm-Šídák test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques:

Journal: JCI Insight

Article Title: Heterozygous NFKB1 variant causes inflammatory dysregulation shaped by broader genetic context in common variable immunodeficiency

doi: 10.1172/jci.insight.198703

Figure Lengend Snippet: ( A ) PCA plot of RNA-seq from iMONO cultures for CA01 and HD with and without heterozygous 1377delT NFKB1 variant. ( B and C ) Volcano plots illustrating distribution of RNA-seq changes from iMONO cultures with and without heterozygous 1377delT NFKB1 variant in CA01 ( B ) and HD backgrounds ( C ). ( D ) Top 10 affected pathways by P value from HALLMARK pathway clustergrams of gene upregulated by heterozygous 1377delT NFKB1 variant in iMONOs from CA01 and HD genetic backgrounds 0, 4, and 6 hours culture with LPS. EMT, epithelial mesenchymal transition. ( E ) Gene expression increased by 1377delT NFKB1 in CA01, but not HD, iMONOs. ( F ) Genes from TNF signaling and inflammatory response gene sets with increased expression due to heterozygous 1377delT NFKB1 in CA01, but not HD, iMONOs. Significant pathways in red. PCA, principal component analysis.

Article Snippet: The primary antibodies used were p105/p50 (Cell Signaling Tech, 3035S), RelB (Cell Signaling Tech, 4954S), C-Rel (Cell Signaling Tech, 4727S), p100/p52 (Cell Signaling Tech, 4882S), and p65 (Cell Signaling Tech, 3035S).

Techniques: RNA Sequencing, Variant Assay, Gene Expression, Expressing

Journal: Cell Death Discovery

Article Title: Epigenetic context defines the transcriptional activity of canonical and noncanonical NF-κB signaling in pancreatic cancer

doi: 10.1038/s41420-026-03019-9

Figure Lengend Snippet: A Western blot analysis of p105/p50, p100/p52, phospho-RELA, and RELB following TNFα and TWEAK treatments across multiple time points (0–30 min and 1–72 h). Representative of n = 3 independent experiments. B Heatmap of differentially expressed genes following RNA-seq analysis of L3.6pl cells treated with TNFα and TWEAK for 6 and 48 hours. Unbiased clustering analysis was performed after differential expression with DESeq2. One TNFα-treated replicate (replicate 1) was excluded due to low read depth and outlier behavior in PCA and z-score heatmap analyses, which indicated a sequencing error. C Venn diagram displaying the number of TNFα-specific, TWEAK-specific, and commonly differentially expressed genes (DEGs), derived from DESeq2 analysis and unbiased clustering analysis. Selections were based on Log2FC ≥ 1, FDR ≤ 0.05, and base mean ≥ 10. D Pathway analysis for biological processes (GO) and KEGG of TNFα-specific (top) and common genes (bottom) identified in ( C ). The top pathways were selected based on FDR values. E Caspase 3/7 activity in L3.6pl and AsPC-1 cells over 72 h following treatment with TNFα (10 ng/ml), TWEAK (10 ng/ml), and cycloheximide (CHX; 10 µM) as indicated. Values represent total green integrated intensity (normalized to vehicle control and cell number at each time point). Area under the curve (AUC) was calculated for each condition, and p -values were determined using unpaired t -tests based on AUC values. n.s. = not significant. F Single-cell migration tracking assay captured every 15 min over 48 h in AsPC-1 and L3.6pl cells using IncuCyte live-cell imaging (10× magnification). Boxplots depict the mean instantaneous speed calculated from migrated tracks for AsPC-1 and L3.6pl cells following vehicle, TNFα, and TWEAK treatments ( p -value: one-way ANOVA; Dunnett’s multiple comparisons relative to vehicle control). G Scatter plot of normalized expression levels of TNFα and TWEAK (log scale) in 173 PDAC patient samples from TCGA. Each dot represents an individual sample. Based on TNFα and TWEAK expression thresholds (TNFα ≥ 100 and TWEAK < 1300), samples were classified as TNFα high (red), TWEAK high (blue; TNFα < 100 and TWEAK ≥ 1300), TNFα/TWEAK low (purple; TNFα < 60 and TWEAK < 700 or not assigned (black). H Gene Set Enrichment Analysis (GSEA) results for TNFα-specific ( n = 310) and common (n = 626; 575 common genes + 51 TWEAK-specific genes) gene sets in the TNFα high , TWEAK high , and TNFα/TWEAK low expression from TCGA data. Normalized Enrichment Scores (NES) and False Discovery Rate (FDR) values are shown in each plot. TNFα high samples show significant enrichment of both TNFα-specific and common gene sets, whereas TWEAK high samples only display enrichment of the TNFα-specific gene set. I Boxplots showing expression of selected genes from TNFα-specific and common gene sets in PDAC samples grouped by TNFα high , TWEAK high , and TNFα/TWEAK low status. Genes were selected based on their rankings from the GSEA analysis. TNFα-specific genes (top panels) show higher expression in TNFα high samples, while common genes (bottom panels) are elevated in both TNFα high and TWEAK high samples. p -values are shown for significant differences (unpaired t-tests).

Article Snippet: The following primary antibodies were used at the indicated dilutions: phospho-NFκB p65 (S536) (93H1) (phospho-RELA; 1:1000; Cell Signaling Technology; 3033; RRID:AB_331284), RELB (D7D7W) (1:1000; Cell Signaling Technology; 10544; RRID:AB_2797727), NF-κB2 p100/p52 (18D10) (1:1000; Cell Signaling Technology; 3017; RRID:AB_10697356), NF-κB1 p105/p50 (D4P4D) (1:1000; Cell Signaling Technology; 13586; RRID:AB_2665516), and β-Actin (D6A8) (1:1000; Cell Signaling Technology; 8457; RRID:AB_10950489).

Techniques: Western Blot, RNA Sequencing, Quantitative Proteomics, Sequencing, Derivative Assay, Activity Assay, Control, Single Cell, Migration, Live Cell Imaging, Expressing