Journal: iScience

Article Title: RNF126 mediates fetal growth restriction via ubiquitination-dependent degradation of the MYH9/MYH10 complex

doi: 10.1016/j.isci.2026.116285

Figure Lengend Snippet: ER Stress mediates the effect of RNF126 on trophoblast function (A) Western blot confirms stable overexpression of Flag-RNF126 (F-RNF126) in BeWo cells. Representative blot is shown from three independent experiments. (B and C) Stable expression of RNF126 inhibited proliferation (B) and promoted apoptosis (C) in BeWo cells. Data are shown as representative images and quantitative analyses ( n = 3 independent experiments). Scale bars, 50 μm. (D and E) The GO and KEGG enrichment analysis plot of the differentially expressed genes (DEGs; |Log2 FoldChange|>0.58, p < 0.05) between two groups. (F) Gene set enrichment analysis of RNA-seq data shows the enrichment of unfolded-protein-response-signaling related genes in OE-RNF126 cells compared to OE-NC cells. (G and H) Representative western blots and quantification show increased GRP78/BiP, pPERK, and CHOP with RNF126 overexpression, and reduced levels upon RNF126 knockdown under TM- or TG-induced ER stress for 24h ( n = 3 independent experiments). (I and J) Representative images and quantification show that 24h treatment with 4-PBA (1 mM) reverses RNF126 overexpression-induced changes in cell proliferation (I) and apoptosis (J) ( n = 3 independent experiments). Scale bars, 50 μm. Data in B, C, H, I, and J are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, n.s. not significant.

Article Snippet: BeWo cells (Procell, Wuhan, China), a human choriocarcinoma-derived trophoblast line commonly used as an in vitro model of human villous trophoblast, were maintained in Ham’s F-12 medium supplemented with 15% fetal bovine serum (Gibco, USA) at 37°C in 5% CO2.

Techniques: Western Blot, Over Expression, Expressing, RNA Sequencing, Knockdown

Journal: iScience

Article Title: RNF126 mediates fetal growth restriction via ubiquitination-dependent degradation of the MYH9/MYH10 complex

doi: 10.1016/j.isci.2026.116285

Figure Lengend Snippet: RNF126 ubiquitinates MYH9 and MYH10, promoting their degradation (A) GST and GST-RNF126 pull-down assays were conducted with BeWo cell lysates (≈1×10 7 cells lysed per pull-down reaction; n = 1 independent experiment). (B and C) MYH9 and MYH10 peptides were identified by LC-MS/MS (using approximately 5∼10 μg of pull-down protein; n = 1 independent experiment). (D and E) Relative protein and mRNA level of RNF126, MYH9, and MYH10 in BeWo cells transfected with control or RNF126 overexpression vectors ( n = 3 independent experiments). (F) Co-IP of MYH9 and MYH10 with RNF126 was conducted in BeWo cells. Representative blot is shown from three independent experiments. (G) Co-IP of Flag-RNF126 with MYH9 and MYH10 was conducted in BeWo cells. Representative blot is shown from three independent experiments. Whole cell lysate was prepared, and immunoprecipitation was performed with Flag beads. (H) Immunofluorescence co-staining of MYH9, MYH10, and RNF126 demonstrates colocalization (Magnification 63×oil, Scale bars, 10 μm, n = 3 independent experiments). (I and J) Representative western blot and densitometric analysis show time-dependent degradation of MYH9 and MYH10 in BeWo cells with or without RNF126 overexpression following cycloheximide (CHX) treatment (0, 2, 4, and 8 h) ( n = 3 independent experiments). (K) MG132 (10 μM), but not CQ (20 μM), restores MYH9 and MYH10 protein levels in RNF126-overexpressing cells after 6 h of treatment. Representative blot is shown from three independent experiments. (L) RNF126 promotes ubiquitination-dependent proteasomal degradation of MYH9 and MYH10. Representative blot is shown from three independent experiments. The error bars represent mean ± SD. ∗ p < 0.05 and ∗∗∗∗ p < 0.0001, n.s. not significant.

Article Snippet: BeWo cells (Procell, Wuhan, China), a human choriocarcinoma-derived trophoblast line commonly used as an in vitro model of human villous trophoblast, were maintained in Ham’s F-12 medium supplemented with 15% fetal bovine serum (Gibco, USA) at 37°C in 5% CO2.

Techniques: Liquid Chromatography with Mass Spectroscopy, Transfection, Control, Over Expression, Co-Immunoprecipitation Assay, Immunoprecipitation, Immunofluorescence, Staining, Western Blot, Ubiquitin Proteomics

Journal: iScience

Article Title: RNF126 mediates fetal growth restriction via ubiquitination-dependent degradation of the MYH9/MYH10 complex

doi: 10.1016/j.isci.2026.116285

Figure Lengend Snippet: RNF126 induces ER stress and trophoblast cell functional alterations by ubiquitinating and degrading the MYH9/MYH10 complex (A) Western blot analysis shows changes in RNF126, MYH9, and MYH10 protein levels after separate overexpression of MYH9 or MYH10 in BeWo cells. Representative blot is shown from three independent experiments. (B) Co-overexpression of MYH9 (1 μg) and MYH10 (1 μg) in RNF126-overexpressing BeWo cells reverses ER stress marker expression more effectively than individual overexpression (2 μg each). Representative blot is shown from three independent experiments. (C and D) The representative graphs and the corresponding quantified histograms demonstrate that co-transfection of MYH9 and MYH10 reverses the effects of cell proliferation and apoptosis caused by RNF126 overexpression ( n = 3 independent experiments). Scale bars, 50 μm. (E) Schematic diagram of serial deletion mutants of Flag-tagged RNF126. (F) Co-IP and western blot analysis show interactions between full-length MYH9-Myc, MYH10-His, and RNF126-Flag deletion mutants in BeWo cell lysates co-transfected with these constructs. Representative blot is shown from three independent experiments. (G) Table listing significantly different ubiquitination sites on MYH9 and MYH10. (H) Western blot analysis of ubiquitination levels of MYH9 and MYH10 in BeWo cells transfected with RNF126-Flag, Ub-HA, and full-length or mutant MYH9/MYH10 constructs. Representative blot is shown from three independent experiments. Data in C and D are presented as mean ± SD. ∗ p < 0.05 and ∗∗∗ p < 0.001, n.s. not significant.

Article Snippet: BeWo cells (Procell, Wuhan, China), a human choriocarcinoma-derived trophoblast line commonly used as an in vitro model of human villous trophoblast, were maintained in Ham’s F-12 medium supplemented with 15% fetal bovine serum (Gibco, USA) at 37°C in 5% CO2.

Techniques: Functional Assay, Western Blot, Over Expression, Marker, Expressing, Cotransfection, Co-Immunoprecipitation Assay, Transfection, Construct, Ubiquitin Proteomics, Mutagenesis

Journal: bioRxiv

Article Title: Real-time, label-free assessment of cell fusion dynamics by high-content imaging

doi: 10.64898/2026.04.08.717136

Figure Lengend Snippet: (A-F) Representative images of BeWo cells stained fbr F-actin (Red) and nuclei (Blue). Control cells (A, C, E) exhibit discrete cell boundaries and cortical actin organization, whereas fbrskolin-treated cells (B, D, F) display loss of distinct borders and formation of multinucleated structures. Scale bars: A, B = 200 μm; C-F = 50 μm. Boxed area in A and B is magnified. (G) Quantification of F-actin fluorescence intensity in control and fbrskolin-treated cells. Data represent mean ± SEM (n = IO images per condition). Statistical significance was assessed using an unpaired two-tailed t-test (***p < 0.001).(H, 1) Relative mRNA expression of CDH1 (H) and ERVW-1 (Syncytin-1) (1) following forskolin treatment. Gene expression was normalized to 18S rRNA and expressed relative to untreated controls. Data represent mean ± SEM from three independent experiments. Statistical significance was assessed using an unpaired two-tailed t-test (*p < 0.05).

Article Snippet: BeWo human choriocarcinoma cells (ATCC) and HeLa cells (ATCC) were cultured in DMEM/F12 (1:1) medium (HiMedia) supplemented with 15% fetal bovine serum and 1% antibiotic– antimycotic solution (Gibco).

Techniques: Staining, Control, Fluorescence, Two Tailed Test, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Real-time, label-free assessment of cell fusion dynamics by high-content imaging

doi: 10.64898/2026.04.08.717136

Figure Lengend Snippet: Representative bright-field time-lapse images of BeWo cells cultured under control (A) or fbrskolin-treated (B) conditions over 48h. (C, D) Automated segmentation and cluster identification using Hannony 5.1 software, with system-assigned pseudocolors indicating distinct detected clusters at the indicated time points. (E, F) Representative digital phase contrast (DPC) images illustrating intracellular texture patterns in control (E) and fbrskolin-treated (F) cells. (G-J) Quantitative time-course analysis of fusion-associated parameters measured at 6 h intervals over 48h: (G) total cluster area (×10 5 μm 2 ). (H) number of detected cell clusters, (I) area-to-cluster ratio (×10 3 ), and (J) cytoplasmic granularity quantified using the SER Bright feature. Control cells are shown in blue and fbrskolin-treated cells in red. Data represent mean ± SEM pooled from three independent experiments (n = 10 images per condition per experiment). Imaging was performed using an Operetta CLS high-content screening system.

Article Snippet: BeWo human choriocarcinoma cells (ATCC) and HeLa cells (ATCC) were cultured in DMEM/F12 (1:1) medium (HiMedia) supplemented with 15% fetal bovine serum and 1% antibiotic– antimycotic solution (Gibco).

Techniques: Cell Culture, Control, Software, Imaging, High Content Screening

Journal: bioRxiv

Article Title: Real-time, label-free assessment of cell fusion dynamics by high-content imaging

doi: 10.64898/2026.04.08.717136

Figure Lengend Snippet: BeWo cells were treated with forskolin (50 μM), a cell-permeable 8-Br-cAMP (cAMP, 1.5 μM), Wortmannin, or their respective combinations, and analyzed using the described imaging and analysis pipeline. (A-C) Quantification of area-to-cluster ratio (A), number of detected clusters (B), and cytoplasmic granularity (C) following treatment with forskolin or cAMP analog relative to untreated controls. (D-F) Corresponding parameters measured following treatment with forskolin alone, Wortmannin alone, or their combination. Data represent mean ± SEM from live-cell imaging experiments (n = 10 images per condition across two independent experiments). Imaging was performed using the Operetta CLS system and analysis was done using Hannony 5.1 software.

Article Snippet: BeWo human choriocarcinoma cells (ATCC) and HeLa cells (ATCC) were cultured in DMEM/F12 (1:1) medium (HiMedia) supplemented with 15% fetal bovine serum and 1% antibiotic– antimycotic solution (Gibco).

Techniques: Imaging, Live Cell Imaging, Software