Journal: Advanced Science

Article Title: De Novo Gene Transcription of Connexin Mediates Cytoplasmic Fluid Exchange and Flocking Transitions in Physiological and Cancerous Epithelial Systems

doi: 10.1002/advs.202508648

Figure Lengend Snippet: De novo transcription is required for the emergence of flocking motion. A–C) Emergence of Flocking motility of HaCaT cells upon EGF treatment in the absence or presence of Actinomycin D (AD) (A,B) or 5, 6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB) (C); DMSO was used as vehicle control for the pharmacological treatment. A) Representative phase contrast image of the maximum intensity projection (MIP) of all frames acquired over a 24‐h period (5 min frame −1 ). Representative images from n = 6 time‐lapse series. Scale bar 100 µM. (B,C) Root Mean Square Velocity (V RMS ) measured by Particle velocimetry analysis (PIV) over 48 h (left) and its mean within the 10–40 h framecut (right). V RMS is expressed as the mean ± SD ( n = 6 independent experiments). D,E) RNAseq analysis of HaCaT cells serum starved for 48h then treated or not with EGF (100ng mL −1 ) for 24 h ( n = 3 independent experiments). D) Differential expression analysis of connexin genes. A comparison of the statistical significance and expression levels of different connexin genes. The left panel (Statistics) displays a bubble plot representing the significance of differentially expressed genes (DEGs), where the size of the dots corresponds to the ‐log10 (adjusted p ‐value), and the color represents the log2 fold change (red indicates upregulation, blue indicates downregulation). The right panel (Expression) shows the absolute expression levels of the same genes as bar plots. Genes such as GJB2 and GJB3 exhibit high differential gene expression. E) Hierarchical clustering heatmap of connexin gene expression. The heatmap represents the expression levels of deregulated connexin genes (GJA1, GJB6, GJB2, GJB3, GJB5, GJB4, GJC1) in control quiescent vs EGF‐treated (for 24 h) HaCat cells. The rows correspond to genes, while the columns represent individual samples. Expression levels are scaled and color‐coded, with red indicating upregulation and blue indicating downregulation. Hierarchical clustering was applied to group genes with similar expression patterns. The top annotation bar denotes the experimental condition (CTRL: yellow, EGF: purple), and the cell type (HaCaT: black). The color scale represents the standardized expression values. F) mRNA levels of Connexins genes in HaCaT cells treated with EGF for 24 h, quantified by qRT‐PCR. Data are the mRNA fold increase relative to the levels of control cells after normalizing for GAPDH and 18S mRNA levels ( n = 6 independent experiments). G) mRNA levels of Connexins genes in HaCaT cells treated with EGF for 0, 1, 6, and 24 h; quantified by qRT‐PCR. Data is the mRNA fold increase relative to the levels of control cells after normalizing for GAPDH and 18S mRNA levels) ( n = 3 independent experiments). H) mRNA levels of Connexin 26 (GJB2) and Connexin 31 (GJB3) treated with low (1 ng mL −1 ) or high (100 ng mL −1 ) doses of EGF for 6 and 24 h. Data are mRNA fold increase relative to the levels of control cells after normalizing for GAPDH and 18S mRNA levels ( n = 4–6 independent experiments). Statistical tests and significance are indicated in Table .

Article Snippet: GJB2 assay ID: Hs00955889_m1.

Techniques: Control, Quantitative Proteomics, Comparison, Expressing, Gene Expression, Quantitative RT-PCR

Journal: Advanced Science

Article Title: De Novo Gene Transcription of Connexin Mediates Cytoplasmic Fluid Exchange and Flocking Transitions in Physiological and Cancerous Epithelial Systems

doi: 10.1002/advs.202508648

Figure Lengend Snippet: Shape variation and intercellular fluid transfer require functional Gap junction channels and specifically Connexins 26 and 31. A–C) HaCaT cells are cultured to confluency, serum starved, and treated with EGF 100 ng mL −1 in the absence or in the presence of Carbonexolone (CBX, 16 µ m ). (A,B) or 18beta‐Glycyrrhetinic acid (GRA, 10 µ m ) (C). A) Representative phase contrast image of the maximum intensity projection (MIP) of all frames acquired over a 24‐h period (5 min frame −1 ). Representative images from n = 5 independent experiments. Scale bar 100 µm. B,C) Root Mean Square Velocity (V RMS ) measured by Particle velocimetry analysis (PIV) over 48h (left) and its mean within cell flocking framecut (right) in control or in cells treated with Connexin inhibitors. V RMS is expressed as the mean ± SD ( n = 3–5 independent experiments). D) Flocking locomotion induced by Amphiregulin (AREG) in Bronchial epithelial primary cultures in Air‐Liquid interface (ALI) in the absence (CTRL) or presence of 50 u m Carbonexolone (CBX). Time evolution of the Root Mean Square Velocity (V RMS ) measured by Particle velocimetry analysis (PIV) over 48 h (left) and its mean within cell flocking framecut (right). V RMS is expressed as the mean ± SD ( n = 3 independent experiments). E,F) Analysis of Aspect/Ratio (AR) (E) and shape index (SI) obtained through automatic cellpose segmentation (F) of HaCaT cells at 0 and 24 h following EGF treatment (100 ng mL −1 ) in the presence of DMSO (EGF), used as vehicle control for the pharmacological treatment, or 16 µ m CBX (EGF‐CBX). ( n = 38–48 fields of view analyzed from 3 independent experiments). G–J) Analysis of intercellular Dye transfer by FRAP (Fluorescence Recovery After Photobleaching) in HaCaT cells loaded with Calcein‐AM (20 µ m , 45 min). The recovery of fluorescence was monitored by acquiring images before (Pre) and after (Post) the recovery. G) Representative images of epithelial cells loaded with a fluorescent dye (Calcein‐AM) in the control condition or treated with 100 ng mL −1 EGF for 24 h. H) Quantification of recovery of fluorescence over 10 min normalized to unbleached area. n = 10–12 fields of view analyzed from 3 independent experiments. I) Representative images of epithelial cells loaded with a fluorescent dye (Calcein‐AM) treated with EGF in the presence of CBX (EGF‐CBX, 16 µ m ) or DMSO EGF (EGF), used as vehicle control. J) Quantification of recovery of fluorescence over 30 min normalized to unbleached area. K,L) Active wetting of FN‐coated substrate of HaCaT spheroids. HaCaT cells were treated with EGF in the presence of CBX (EGF‐CBX, 100 µM) or DMSO (EGF), used as a control. K) Representative phase contrast images of spheroids at different timepoints during active wetting. The spheroid contour is indicated by a dotted line. ( n = 3 independent experiments). Scale bar 500 µ m . L) Quantification of the spreading area at different timepoints normalized to T0h. M,N) Collective flocking motility induced by EGF treatment in HaCaT clones following depletion of Connexin 26, Connexin 31, or both (Cx26KO, Cx31KO, and Cx26/31KO respectively). Time evolution of the Root Mean Square Velocity (V RMS ) measured by Particle velocimetry analysis (PIV) over 48 h (left) and its mean within the 10–40 h framecut (right). V RMS is expressed as the mean ± SD ( n = 3–6 independent experiments). Statistical tests and significance are indicated in Table .

Article Snippet: GJB2 assay ID: Hs00955889_m1.

Techniques: Functional Assay, Cell Culture, Control, Fluorescence, Clone Assay