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: GJB3 assay ID: Hs02378125_s1.

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: 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: GJB4 assay ID: Hs00920816_s1.

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

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Bioengineering-tissue strategies to model mammalian implantation in vitro

doi: 10.3389/fbioe.2024.1430235

Figure Lengend Snippet: List of primers used for quantitative PCR analysis.

Article Snippet: ZO1 , Tight Junction Protein ZO-1 , Ss03373514_m1.

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Bioengineering-tissue strategies to model mammalian implantation in vitro

doi: 10.3389/fbioe.2024.1430235

Figure Lengend Snippet: Generation of the porcine 3D endometrial model and its characterization. (A) Schematic representation of the 3D endometrial model assembling, from uterine biopsy to endometrial epithelial cell and stromal fibroblast isolation and culture onto 2D standard plastic dishes (scale bar 100 μm). (B) Hematoxylin and Eosin staining of the 3D endometrial model obtained by co-culturing epithelial cells and stromal fibroblasts onto highly porous polystyrene scaffolds (scale bars 100 and 50 μm). (C) Transcription levels of VIM, COL3A1, KRT18, ZO1, CDH1 and EPCAM genes in endometrial stromal cells cultured on 2D culture systems (blue bars), endometrial epithelial cells cultured on 2D culture systems (pink bar), 3D endometrial models (yellow bars) and in vivo endometrial tissue, as a positive control (green bars). Gene expression is presented with the highest level set to 1 and all others relative to this. Data are expressed as the mean ± the standard error of the mean (SEM). a,b Different superscripts indicate p < 0.05. ND: not detected. (D) Immunofluorescent staining of 3D endometrial models for VIM (red) and ZO1 (green). Nuclei are counterstained with DAPI (blue) (scale bars 100 and 20 μm).

Article Snippet: ZO1 , Tight Junction Protein ZO-1 , Ss03373514_m1.

Techniques: Isolation, Staining, Cell Culture, In Vivo, Positive Control, Gene Expression

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Bioengineering-tissue strategies to model mammalian implantation in vitro

doi: 10.3389/fbioe.2024.1430235

Figure Lengend Snippet: Representative image of a TR spheroid attached to the 3D endometrial model. (A) Hematoxylin and Eosin staining of a TR spheroid adhering to the 3D endometrial culture system after a 24-h co-culture. No space between the spheroid and the epithelial compartment was visible (scale bar 50 μm). (B) Immunohistochemical co-staining for the mature TR marker GATA3 (red) and ZO1 (green) of a TR spheroid adhering to the 3D endometrial culture system. Nuclei were counterstained with DAPI (blue) (scale bars 100 μm).

Article Snippet: ZO1 , Tight Junction Protein ZO-1 , Ss03373514_m1.

Techniques: Staining, Co-Culture Assay, Immunohistochemical staining, Marker