Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: A) Schematic for domain structures of Ect2 wild-type (Ect2-wt), N-terminally truncated active (ΔN-Ect2) and the catalytically inactive variant with PVQR->AAAA (564-567) substitutions (ΔN-Ect2-DHmut). B) Representative maximum intensity projections of 3D-SIM images of U2OS cells expressing EGFP control or the indicated EGFP-tagged Ect2 constructs. Upper panels show EGFP or EGFP-tagged Ect2 localization, middle panels display F-actin stained with phalloidin, and lower panels present magnified views of the regions depicted in the actin images. Scale bar: 10 µm. n = 7-15 cells. C) Quantification of mean phalloidin fluorescence intensity in wide-field images of cells expressing the indicated constructs. Cells were stained with WGA to visualize cell morphology and with phalloidin to label F-actin. Transfected cells were identified EGFP fluorescence. F-actin intensity was quantified by automated image analysis as described in the Methods. Data represent n ≥ 276 cells from 4 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Bars indicate mean ± SEM. D and H) Representative TIRF images of the Rho activity sensor (mCherry-Rhotekin RBD) co-expressing either EGFP control, constitutively active Ect2 (EGFP-ΔN-Ect2) (D) or constitutively active GEF-H1 C53R (H) . Two phenotypes induced by active Ect2 are shown in (D) : (top) reduced pulsatory Rho sensor signal dynamics and (bottom) peripheral enrichment of Rho sensor signal with slow circumferential movement (white arrow). Frame rate: 3 frames/min, scale bar, 20 µm. E and F) Percent cells with peripheral Rho sensor enrichment (E) and the frequency of Rho sensor pulses in the central cell region (F) ; n ≥ 32 cells from 4 independent experiments. (G) Average pulse frequency of the Rho activity sensor signal upon co-expression of EGFP-control, active EGFP-ΔN-Ect2 or a mutant which cannot bind to active Rho (EGFP-ΔN-Ect2-RBmut); n ≥ 25 cells from 3 independent experiments. Bars indicate mean ± SEM.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Variant Assay, Expressing, Control, Construct, Staining, Fluorescence, Transfection, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: Background-corrected average EGFP intensity in U2OS cells expressing EGFP control (black dots) or EGFP-ΔN-Ect2 (green dots), plotted against the normalized mean pulse frequency of the RBD sensor. Each dot represents a single cell. Data are from n ≥52 cells across three independent experiments.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Expressing, Control, Single Cell

Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: A) Representative spinning disk confocal images of fixed U2OS cells transfected with either EGFP control, active Ect2 (EGFP-ΔN-Ect2), or a variant lacking the C-terminal PBC region (EGFP-ΔN-Ect2-ΔPBC). A schematic of the constructs is shown in . Upper panels: single central z-plane, lower panels: side views show orthogonal (x-z) projections of the z-stack along the line indicated in the corresponding upper panels. Scale bars: 10 µm (xy), 5 µm (z), n=50-56 cells from 3 independent experiments. B) Average pulse frequency of the Rho activity sensor signal (mCherry-Rhotekin-RBD) in cells expressing the indicated Ect2 variants. Red dots mark cells that generate fast high-amplitude pulses. n ≥ 51 cells from 3 independent experiments. using one-way ANOVA followed by Tukey’s post hoc test. Bars indicate mean ± SEM. C, E, G) Representative TIRF images of cells expressing the Rho activity sensor (right) and the indicated constructs (left), respectively. Frame rate: 3 frames/min, scale bar = 20 µm. D, F, H) Mean normalized intensity values of the Rho sensor signal in the corresponding cell regions (white boxes) in (C, E, F) . I) Proposed interplay between Ect2 and pulsatory Rho contraction signal network dynamics. Left: Pulsatory Rho contraction depends on a positive feedback loop in which Lbc-GEFs are recruited to the plasma membrane through binding to active Rho (right angled blue arrow). Curved red arrow points to enzymatic activation. Middle: In contrast, Ect2 is constitutively associated with the plasma membrane, independently of active Rho . Increased local concentration of Ect2 may result in a higher effective on-rate, thereby conferring a kinetic advantage that can outcompete Lbc-GEFs and thereby suppress Rho activity pulses. Curved red arrows illustrate enzymatic activation and allosteric activation. Right: Loss of the C-terminal PBC reduces Ect2 plasma membrane association, shifting its properties towards a Lbc-GEF-like phenotype, that is based on plasma membrane recruitment to active Rho, and that stimulates Rho activity pulses.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Transfection, Control, Variant Assay, Construct, Activity Assay, Expressing, Clinical Proteomics, Membrane, Binding Assay, Activation Assay, Concentration Assay

Journal: Chembiochem

Article Title: Monoclonal Antibodies Accessing the Cytosol of Living Cells and Binding to Polo‐Like Kinase 1 Interdomain Linker Affect Mitotic Behavior

doi: 10.1002/cbic.202500858

Figure Lengend Snippet: Anti‐PLK1 mAbs characterization. (A) Clones 35‐206, 3F8, and 13E8 were raised against full‐length PLK1, fragment 300–600, or fragment 300–400, respectively. KD: Kinase Domain; IDL: Interdomain Linker; PBD: Polo Box Domain. (B) Epitope mapping by Western blot. Lane 1: protein ladder; Lanes 2‐4: U2OS cells expressing 68 kDa PLK1, 14 kDa IDL (300–400), or 35 kDa IDL‐PBD (300–603), respectively.

Article Snippet: Human cervical carcinoma cells HeLa (ATCC Cat# CCL‐2, RRID:CVCL_0030), histone‐green fluorescent protein expressing HeLa cells H2BGFP‐HeLa (SCC117, Merck Millipore, RRID:CVCL_ZM02), human osteosarcoma cells U2OS (ATCC HTB‐96) U2OS (RRID:CVCL_0042) and human osteosarcoma cells expressing both luciferase (LUC) and green fluorescent protein (EGFP) (EGFPLuc‐U2OS, produced in our laboratory) were cultured adherently on plastic substrates (75 cm 2 Falcon tissue culture flasks) in high‐glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Perbio, Brebières, France), 2 mM L‐glutamine, 100 U/mL penicillin G, and 100 μg/mL streptomycin.

Techniques: Clone Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and C ) Representative microscopic images of BrdU incorporation assays in U2OS and MG63 osteosarcoma cells under normoxic (control) and CoCl 2 induced hypoxic conditions. Scale bar = 50 μm. ( B and D ) Quantification of BrdU-positive cells showing a significant decrease in proliferation in hypoxic U2OS and MG63 cells compared with their respective controls (n ≥ 20 cells per condition). ( E and G ) Representative images of colony-formation assays in control and CoCl₂-treated hypoxic U2OS and MG63 cells, respectively. (F and H) Quantification of colony numbers showing a marked reduction in the clonogenic potential of hypoxic osteosarcoma cells. ( I and K ). Representative images of cell-migration assays in U2OS and MG63 cells under control and hypoxic conditions. Scale bar = 50 µm. ( J and L) Quantification of migrated cells showed a significant reduction in the migratory capacity of hypoxic U2OS and MG63 cells, respectively. Statistical significance was calculated using two-tailed Student’s t -test and is represented as mean ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: BrdU Incorporation Assay, Control, Migration, Two Tailed Test

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A ) U2OS cells, either stably expressing K294A upon doxycycline induction or uninduced controls, were biochemically fractionated into nuclear and cytoplasmic compartments. Western blot analysis confirmed fractionation quality using Lamin A/C as a nuclear marker and GAPDH as a cytoplasmic marker. Myc blotting verified the expression of K294A upon doxycycline induction. ( B ) Quantitative real-time PCR analysis revealed a significant reduction in the cytoplasmic levels of RORA and KCTD16 transcripts in K294A expressing cells compared with controls, while BNIP3 levels remained unchanged. ( C ) Western blot analysis further confirmed decreased protein levels of RORA and KCTD16 in K294A-expressing cells. Myc blotting verified stable K294A expression. ( D ) Densitometric analysis of RORA and KCTD16 protein bands from panel C was performed using ImageJ software. β-Actin was used as a loading control for normalization. Statistical analysis was calculated by performing two-tailed Student’s t -test. ( E ) Table summarizing the internal CAGE (Cap Analysis of Gene Expression) sites identified within the analysed transcripts, with the specific positions highlighted in red. ( F - J ) Bar graphs representing the genomic distribution of CAGE peaks for each gene, illustrating the relative frequency of CAGE signals across different transcript regions. ( K ) Schematic illustration of the Xrn1 susceptibility assay used to assess the stability of 5′-capped transcripts. ( L ) Relative 5′-end loss of RORA and KCTD16 was assessed using an in vitro Xrn1 susceptibility assay. In K294A-expressing cells, both transcripts exhibited a level of 5′-end loss comparable to STAT3 , a known cCE target, relative to control cells. Statistical analysis was performed using one sample Student’s t -test. ( M ) Western blot analysis showing Xrn1 protein levels in Xrn1 knockdown cells with or without doxycycline-induced K294A expression. Myc detection confirmed successful induction of the dominant-negative cCE mutant. ( N ) Quantification of Xrn1 knockdown efficiency was performed using ImageJ software, with β-Actin serving as the internal loading control. ( O ) RORA and KCTD16 exhibited the most pronounced rescue in Xrn1 knockdown cells expressing K294A, indicating their strong dependence on cytoplasmic capping for stability. Statistical significance was determined using one-way ANOVA. All the data is represented as mean ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Stable Transfection, Expressing, Western Blot, Fractionation, Marker, Real-time Polymerase Chain Reaction, Software, Control, Two Tailed Test, Gene Expression, Drug Susceptibility Assay, In Vitro, Knockdown, Dominant Negative Mutation, Mutagenesis

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and B ) qPCR analysis of the selected transcripts in U2OS and MG63 cells revealed reduced expression following treatment with the HIF1α inhibitor PX478, irrespective of hypoxia induction. ( C and E ) Western blot analysis of U2OS and MG63 cells demonstrated reduced protein levels of all selected targets, including HIF1α, in PX478-treated hypoxic samples. ( D and F ) Quantification of western blot band intensities corresponding to panels C and E was performed using ImageJ software. β-Actin served as the loading control for normalization. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined using one-way ANOVA. ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Expressing, Western Blot, Software, Control

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A ) Western blot analysis of c-Myc protein levels in U2OS cells under normoxic and CoCl₂-induced hypoxic conditions. ( B ) Densitometric quantification of c-Myc expression from panel A using ImageJ, showing reduced c-Myc levels in hypoxic U2OS cells. β-Actin served as the loading control. Statistical significance was determined using two-tailed Student’s t -test. ( C and E ) Western blots showing siRNA-mediated depletion of RORA ( C ) and KCTD16 ( I ) in U2OS and MG63 cells under hypoxic conditions. HIF1α blot confirms hypoxia induction. c-Myc levels were elevated upon depletion of either gene. ( D and J ) ImageJ-based densitometric quantification of blots from panels C and I , normalized to β-actin. Statistical analysis was performed using one-way ANOVA. ( E and K ) Representative microscopic images of BrdU incorporation assays in RORA and KCTD16 depleted hypoxic U2OS and MG63 cells, respectively. Scale bar = 50 μm. ( F and L ) Quantification of BrdU-positive cells showing increased proliferation upon RORA or KCTD16 depletion under hypoxic conditions (n ≥ 20 cells per condition). ( G and M ) Representative images of colony formation assays in RORA-and KCTD16-depleted hypoxic osteosarcoma cells. ( H and N ) Quantitative analysis showing enhanced clonogenic potential following RORA or KCTD16 depletion in hypoxic cells. Statistical significance was calculated using one-way ANOVA. All the data is represented as ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Western Blot, Expressing, Control, Two Tailed Test, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and I ) Western blots showing RORA, KCTD16, and c-Myc levels in U2OS and MG63 cells, respectively. ( B and J ) Densitometric quantification of the blots using ImageJ demonstrated that overexpression of RORA or KCTD16 led to reduced c-Myc expression in both cell lines. β-Actin was used as a loading control for normalization. ( C and K ) Representative microscopic images of BrdU incorporation assays in RORA and KCTD16 overexpressing U2OS and MG63 cells, respectively. Scale bar = 50 μm. ( D and L ) Quantification of BrdU-positive cells showing significantly decreased proliferative capacity in RORA and KCTD16-overexpressing cells (n ≥ 20 cells per condition). ( E and M ) Representative images of colony formation assays in RORA and KCTD16 overexpressing osteosarcoma cells. ( F and N ) Quantification of colonies demonstrating a marked reduction in clonogenic potential upon RORA or KCTD16 overexpression. ( G and O ) Representative images of migration assays in RORA and KCTD16-overexpressing cells. Scale bar = 50 μm. ( H and P ) Quantification of migrated cells showing significantly impaired migratory capacity in RORA and KCTD16 overexpressing osteosarcoma cells. Statistical analysis was performed using one-way ANOVA, and all data are presented as mean ± SD from three independent biological replicates. Significance is indicated as follows: ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Western Blot, Over Expression, Expressing, Control, BrdU Incorporation Assay, Migration