Journal: iScience

Article Title: Single-cell transcriptomic landscape of keratinocyte transformation from actinic keratosis to skin carcinoma

doi: 10.1016/j.isci.2025.114169

Figure Lengend Snippet: Functional validation of IL-13 signaling in keratinocytes (A) Activity scores of the top 10 prioritized ligands expressed in other cell types and their corresponding receptors expressed in Ker-6 ( KRT6A + KRT16 + ) keratinocytes (top). Predicted ligand-receptor interaction potentials involving Ker-6 ( KRT6A + KRT16 + ) keratinocytes (bottom). (B) Immunofluorescence staining for KRT6A/HMGB2 (top) and KRT16/HMGB2 (bottom) in NS, AK, and cSCC samples. Scale bars, 100 μm. (C) Line plots showing the relative cell numbers of HaCaT (top) and HSC-5 (bottom) cells after IL-4/IL-13 inhibitor treatment compared to controls. (D) Quantitative RT-PCR analysis confirming efficient knockdown (>70%) of IL13RA1 and IL4R expression in HaCaT and HSC-5 cells following lentiviral shRNA transduction. Data are presented as mean ± SEM from independent experiments ( n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t test, comparing each knockdown group to its respective negative control (NC) group. ∗∗ p < 0.01. (E) CCK-8 proliferation assays showing time-dependent growth curves of control and receptor-silenced cells under basal conditions or after IL-13 stimulation. Data are presented as mean ± SEM from independent experiments ( n = 5). Statistical significance was determined using pairwise t tests. ∗ p < 0.05 and ∗∗ p < 0.01 indicate significant differences between the indicated knockdown group and the NC group under the same condition (with or without IL-13) at the respective time point. (F) Representative transwell migration images illustrating reduced motility of IL13RA1- or IL4R-deficient HaCaT cells compared with negative controls. (G) Representative transwell migration images illustrating reduced motility of IL13RA1- or IL4R-deficient HSC-5 cells compared with negative controls. For quantification in (F) and (G), cells were seeded in the upper chamber at a density of 3 × 10 5 cells per well in a 24-well plate format and allowed to migrate for an appropriate duration. (H) Western blot analysis of Bcl-xL and KRT17 expression in control and knockdown cells, with or without IL-13 treatment, demonstrating suppression of survival and proliferation markers upon receptor silencing.

Article Snippet: Rabbit anti-IL4R antibody , Cell Signaling Technology , Cat# 8720; RRID: AB_10829029.

Techniques: Functional Assay, Biomarker Discovery, Activity Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Knockdown, Expressing, shRNA, Transduction, Two Tailed Test, Negative Control, CCK-8 Assay, Control, Migration, Western Blot

Journal: bioRxiv

Article Title: Chromosome condensation mechanically primes the nucleus for mitosis

doi: 10.1101/2025.09.01.673438

Figure Lengend Snippet: A) Scheme with the multiple strategies employed to interfere with mitotic chromosome structure. B) Quantification of the orientation bias in NE fluctuations for control cells (DMSO; n=30) and cells treated with either ICRF-193 (n=29; p<0.0481) or VPA (n=27; p<0.0461). Values above zero reflect a bias towards inwards fluctuations. C) Representative frames from movies of cells expressing cyclin B1-Venus/H2B-RFP treated with DMSO (top panel), ICRF-193 (middle panel) or VPA (bottom panel). Time is represented in minutes (min) and zero corresponds to the moment of CDK1i washout. D) Quantification of the time for cyclin B1 nuclear translocation after CDK1i washout to for the indicated treatments (n=43 for DMSO; n=38 for ICRF-193; n=43 for VPA; p<0.0001). E) Cumulative percentage of cells with cyclin B1 nuclear translocation following CDK1i washout. F) Representative frames of HeLa cells expressing DHC-GFP cells during mitotic entry, after treatment with DMSO (top panel) or ICRF-193 (bottom panel). Yellow arrowheads indicate the NE. Right panels show kymographs highlighting the absence of dynein on the NE of ICRF-193 treated cells. Horizontal scale bar, 10 μm. Vertical scale bar, 200 sec. G) Representative immunofluorescence images of prophase cells expressing DHC-GFP after treatment with DMSO, ICRF-193 or VPA. H) Quantification of the levels of DHC-GFP fluorescence intensity on the NE for DMSO (n=52), ICRF-193 (n=63) or VPA (n=49) treated cells (p<0.0001). I) Representative immunofluorescence images of DHC-GFP localization at the NE, following depletion of SMC2 by RNAi. J) Quantification of the levels of DHC-GFP on the NE following for control cells (n=74) or SMC2-depleted cells (SMC2 RNAi; n= 33; p<0.0001). Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used: mouse anti-Lamin A/C (1:500, Abcam), rabbit anti-Lamin B1 (1:500, Abcam), rat anti-alpha tubulin (1:500, Bio-Rad), rabbit anti-NudE/NudEL antibody (1:500, gift from Richard Vallee), rabbit anti-BicD2 (1:500, Atlas Antibodies) rabbit anti-SUN1 (1:1000, Sigma-Aldrich), rabbit anti-SUN2 (1:1000, Sigma-Aldrich), rabbit anti-SMC2 antibody (1:500; Bethyl Laboratories) rabbit anti-cPLA2 (1:100, #2832; Cell Signaling), rabbit anti-CENP-F (ab5, 1:300; Abcam).

Techniques: Control, Expressing, Translocation Assay, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Chromosome condensation mechanically primes the nucleus for mitosis

doi: 10.1101/2025.09.01.673438

Figure Lengend Snippet: A) Representative immunofluorescence images of the NE of prophase cells following disruption of chromosome structure. B) Quantification of the excess of perimeter (EOP) in nuclei of interphase (n=59) and prophase (n=56) cells treated with DMSO, as well as prophase cells treated with ICRF-193 (n=56) or VPA (n=49; p<0.0001). C) Representative immunofluorescence images of nuclei stained for cPLA2 in control cells (DMSO, n=53), and cells treated with ICRF-193 (n=52) or VPA (n=46) in prophase. D) Quantification of cPLA2 levels in the NE and nucleus of DMSO, ICRF-193 (p<0.0001) or VPA (p<0.0001) treated prophase cells. E) Representative immunofluorescence images of nuclei stained for cPLA2 in control (scramble RNAi; n=37) or SMC2 depleted cells (SMC2 RNAi; n=39). F) Quantification of the EOP in nuclei of prophase cells treated with control or SMC2 RNAi (p<0.0001). G) Quantification cPLA2 in the NE (p<0.0001) and nucleus (p=0.0028) of cells depleted of SMC2 by RNAi. H) Representative TEM images of the NE of cells treated with ICRF-193 in interphase (left panel) and prophase (right panel), highlighting the NE in blue and NPCs in yellow. Quantification of the nuclear pore size (I) and perinuclear space (J) in interphase (n= 6 cells; 67 NPCs) and prophase cells treated with ICRF-193 (n=17 cells; 168 NPCs). Scale bars in immunofluorescence images, 10 μm. Scale bars in TEM images, 100 nm.

Article Snippet: The following primary antibodies were used: mouse anti-Lamin A/C (1:500, Abcam), rabbit anti-Lamin B1 (1:500, Abcam), rat anti-alpha tubulin (1:500, Bio-Rad), rabbit anti-NudE/NudEL antibody (1:500, gift from Richard Vallee), rabbit anti-BicD2 (1:500, Atlas Antibodies) rabbit anti-SUN1 (1:1000, Sigma-Aldrich), rabbit anti-SUN2 (1:1000, Sigma-Aldrich), rabbit anti-SMC2 antibody (1:500; Bethyl Laboratories) rabbit anti-cPLA2 (1:100, #2832; Cell Signaling), rabbit anti-CENP-F (ab5, 1:300; Abcam).

Techniques: Immunofluorescence, Disruption, Staining, Control, Pore Size

Journal: bioRxiv

Article Title: Chromosome condensation mechanically primes the nucleus for mitosis

doi: 10.1101/2025.09.01.673438

Figure Lengend Snippet: A) Schematic representation of the experimental setup designed to assess the response of the prophase nucleus to mechanical stimulation. B) Representative images of control HeLa cell in prophase expressing DHC-GFP and stained with SiR-DNA under mechanical stimulation. Time zero corresponds to the moment when compression was applied to the nucleus. C) Ratio between the intensity of dynein on the NE and cytoplasm for control prophase cells (n=12). Note how the ratio is above 1, reflecting an enrichment of dynein on the NE during this stage of the cell cycle. D) Representative images of a prophase cell treated with ICRF-193 expressing DHC-GFP and stained with SiR-DNA, under mechanical stimulation. Time zero corresponds to the moment when compression was applied to the nucleus. E) Ratio between the intensity of dynein on the NE and cytoplasm for prophase cells treated with ICRF-193 (n=12). Note how the ratio increases following mechanical stimulation. F) Representative immunofluorescence images of DMSO (n=48), ICRF-193 (n=58) or VPA (n=22) treated cells after confinement, showing localization dynein on the NE. G) Quantification of DHC-GFP levels on the NE after mechanical stimulation. Note how ICRF-193 and VPA-treated cells accumulate dynein on the NE, similar to controls. H) Representative immunofluorescence images of controls (Scramble RNAi) and SMC2 depleted cells (SMC2 RNAi) showing dynein localization on the NE. I) Quantification of DHC-GFP levels on the NE of control cells (n=28) and cells depleted of SMC2 (n=23) after mechanical stimulation. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used: mouse anti-Lamin A/C (1:500, Abcam), rabbit anti-Lamin B1 (1:500, Abcam), rat anti-alpha tubulin (1:500, Bio-Rad), rabbit anti-NudE/NudEL antibody (1:500, gift from Richard Vallee), rabbit anti-BicD2 (1:500, Atlas Antibodies) rabbit anti-SUN1 (1:1000, Sigma-Aldrich), rabbit anti-SUN2 (1:1000, Sigma-Aldrich), rabbit anti-SMC2 antibody (1:500; Bethyl Laboratories) rabbit anti-cPLA2 (1:100, #2832; Cell Signaling), rabbit anti-CENP-F (ab5, 1:300; Abcam).

Techniques: Control, Expressing, Staining, Immunofluorescence