Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Inhibition of branched actin leads to increased endosome size. (A-J) . HeLa cells were ( A-C ) untreated, ( D-E ) treated with the formin inhibitor SMIFH2 (25 µM), ( F-G ) treated with the inactive control CK-689 (300 µM), or ( H-J ) the ARP2/3 branched actin inhibitor CK-666 (300 µM) for 50 min. Cells were fixed and stained with EEA1 and cortactin. Merged images (panels C and J ) show decreased cortactin at endosomes upon CK-666 treatment. (K) . Quantification of the effects of the inhibitors on endosome size as depicted in A-J. Imaris software was used to render EEA1-decorated endosomes as surfaces, and endosome size for each treatment (µm 2 ) was normalized to the average endosome size of the untreated group. Quantification represents 30 images from three independent experiments, including ∼50,000 endosomes per treatment. A two-tailed Mann-Whitney nonparametric test was used to determine p -values between treatment groups.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Control, Staining, Software, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Transferrin recycling is impaired upon ARP2/3 inhibition. (A-D) . HeLa cells on coverslips were incubated with fluorophore-labeled transferrin (Tf-488) for 10 minutes of uptake. Cells were ( A ) untreated, ( B ) treated with 25 µM SMIFH2, ( C ) treated with 300 µM CK-689, or ( D ) treated with 300 µM CK-666 during the transferrin uptake. Images are representative of a treated coverslip after uptake. (E-H) . Cells were chased in ( E ) complete media, ( F ) complete media containing 25 µM SMIFH2, ( G ) complete media containing 300 µM CK-689, or ( H ) complete media containing 300 µM CK-666 for 40 minutes to allow recycling. Images are representative of cells on an treated coverslip after chase. (I) . The arithmetic mean intensity of each image was analyzed using Zeiss Zen Blue software after uptake and chase. For each treatment condition, the internalized mean for the uptake was set at 100%, and the arithmetic mean intensity for each recycling image was expressed as a percentage of the normalized uptake for that condition. Quantification represents 30 images from three independent experiments. Statistical significance was determined using a two-tailed unpaired t -test.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Incubation, Labeling, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Transferrin and EGF are delayed at early endosomes upon ARP2/3 inhibition. (A-D) . HeLa cells were incubated with Tf-488 diluted in complete media for 10 min. Following uptake, cells were chased in ( A-B ) complete media or ( C-D ) in media containing 300 µM CK-666. Representative images are from the 30 min chase time point. (E). The percentage of Tf-488 fluorescence in EEA1 endosomes was calculated by measuring the area of Tf-EEA1 overlap as a percentage of total Tf fluorescence area. Statistical analysis was performed between the untreated and CK-666-treated groups at each time point. Quantification represents 24 images from three independent experiments. Statistical significance for the 30 min and 45 min time points was determined using a two-tailed Mann-Whitney nonparametric test, and an unpaired two-tailed t -test was used for the 15 min time point. (F). A bar graph showing the individual data points from the 30 min time point in ( E ). (G-J) . HeLa cells were serum starved for 1 h, then incubated with EGF-488 diluted in complete media for 10 min. Following uptake, cells were chased in ( G-H ) complete media or ( I-J ) in complete media containing 300 µM CK-666. Representative images are from the 45 min time point of media chase. (K). The percentage of EGF-488 fluorescence in EEA1-marked endosomes was calculated by measuring the area of EGF-EEA1 overlap as a percentage of total EGF fluorescence area. Statistical analysis was performed between the untreated and CK-666-treated groups at each time point. Quantification represents 24 images from three independent experiments. Statistical significance for the 15 min and 45 min time points was determined using a two-tailed Mann-Whitney nonparametric test, and an unpaired two-tailed t -test was used for the 30 min time point. (L). A bar graph showing the individual data points from the 45 min time point in ( K ).

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Incubation, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Branched actin inhibition, but not formin inhibition, decreases actin at RAB5 QL endosomes. (A-P) . HeLa cells were transfected with mCherry-RAB5 Q79L and were either ( A-D ) untreated or treated with ( E-H ) 25 µM SMIFH2, ( I-L ) 300 µM CK-689, or ( M-P ) 300 µM CK-666 for 20 min. Cells were fixed and co-stained with cortactin and phalloidin to visualize the actin network. (Q) . Quantification of ( A-P ). RAB5 Q79L endosomes that colocalized with phalloidin or cortactin were counted as a percentage of total RAB5 Q79L endosomes. Quantification represents 15 images from three independent experiments. A two-tailed Mann-Whitney nonparametric test was used to determine significance between treatment groups. Data comparisons without error bars are not significant ( p > 0.05).

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Transfection, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Transferrin is bounded by cortactin at endosomes. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) 25 µM SMIFH2, ( G-I ) 300 µM CK-689, or ( J-L ) 300 µM CK-666 for 4 min. Following the pre-treatment, cells were incubated with Tf-488 (and their respective inhibitor) for 6 min of uptake. (M-P) . Representative fluorescence intensity profiles of endosomes from ( M ) untreated, ( N ) SMIFH2-treated cells, ( O ) CK-689-treated cells, or ( P ) CK-666-treated cells. The intensity profile of Tf is in green, and the intensity profile of cortactin is in magenta. Tf vertices above 130% that occur within 20 degrees of a cortactin value above 130% are represented with a red circle; these peaks are “bounded”. Tf vertices above 130% that are not within 20 degrees of a cortactin value above 130% are represented with a grey circle and are “not bounded”. (Q). The percentage of “bounded” Tf peaks was quantified from fluorescence intensity profiles (as represented in ( M-P )). Quantification is from three independent experiments, and from 37 endosomes for the untreated group, 43 endosomes for SMIFH2-treated, 38 endosomes for CK-689-treated, and 37 endosomes from the CK-666-treated group. Statistical significance was determined using a two-tailed unpaired t -test (ns: p > 0.05). (R, S) . Representative models for the quantification and results of the experiment. A circle was drawn around the endosome membrane (red) that intersects with the regions of Tf (green) and cortactin (magenta), and fluorescence intensity at each degree around the circle was measured. ( R ) Untreated cells have Tf in confined regions on the endosome and are adjacent to regions of cortactin ∼60% of the time. ( S ) CK-666-treated cells have regions of Tf that are broader and “not bounded” by cortactin.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Transfection, Incubation, Fluorescence, Two Tailed Test, Membrane

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Tf occupies less discrete regions on the endosome when branched actin is inhibited. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) 25 µM SMIFH2, ( G-I ) 300 µM CK-689, or ( J-L ) 300 µM CK-666 for 4 min. Following the pre-treatment, cells were incubated with Tf-488 (and their respective inhibitor) for 6 min of uptake. Untreated, SMIFH2-treated, and CK-689-treated cells show Tf localized to discrete regions on the endosome (yellow arrows). CK-666-treated cells have broader regions of Tf on the endosome (blue arrows). (M). Model for quantification. A circle (blue) was drawn around the endosome membrane (red) that intersects with the regions of Tf, and the fluorescence intensity at each degree around the circle was measured. (N-Q) . Representative fluorescence intensity profiles of Tf on the endosome from ( N ) untreated, ( O ) SMIFH2-treated, ( P ) CK-689-treated, and ( Q ) CK-666-treated cells. The profiles depicted are from the endosomes indicated with a magenta-colored star ( A-L ). (R). Tf-containing regions from the fluorescence intensity profiles ( N-Q ) were identified, and the fluorescence values ± 20 degrees from the maximum were normalized. These normalized profiles of Tf-containing regions are Tf “peaks”. The graph shows the average profile of 72 Tf peaks from 37 endosomes for the control group, 70 peaks from 43 endosomes from SMIFH2-treated cells, 73 peaks from 38 endosomes from CK-689-treated cells, and 70 peaks from 37 endosomes from CK-666-treated cells. Data are from three independent experiments. The average profile of Tf peaks in the CK-666-treated cells is broader (less discrete) than the other treatment groups. See table in Figure EV3 for statistical information.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Transfection, Incubation, Membrane, Fluorescence, Control

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: EGF segregation on the endosome is affected by branched actin inhibition. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) SMIFH2, ( G-I ) CK-689, or ( J-L ) CK-666 for 4 min. Following the pre-treatment, cells were incubated with EGF-488 (and the respective inhibitor) for 17 min of uptake. Untreated, SMIFH2-treated, and CK-689-treated cells show EGF localized to discrete regions on the endosome (yellow arrows). CK-666-treated cells have broader regions of EGF on the endosome (blue arrows). (M-P) . Representative fluorescence intensity profiles of EGF on the endosome from the ( M ) untreated, ( N ) SMIFH2-treated, ( O ) CK-689-treated, and ( P ) CK-666 treated cells. The profiles depicted are from the endosomes indicated with a magenta star in ( A-L ). (Q) . EGF-containing regions from the fluorescence intensity profiles ( M-P ) were identified, and the fluorescence values ± 20 degrees from the maximum were normalized. These normalized profiles of EGF-containing regions are EGF “peaks”. The graph shows the average profile of 84 EGF peaks from 47 endosomes that were analyzed for the control group, 86 peaks from 51 endosomes from SMIFH2-treated cells, 72 peaks from 39 endosomes from CK-689-treated cells, and 86 peaks from 49 endosomes from CK-666-treated cells. Data are from three independent experiments. The average profile of EGF peaks in the CK-666-treated cells is broader (less discrete) than the other treatment groups. See table in Figure EV4 for statistical information.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Transfection, Incubation, Fluorescence, Control

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: The degradative and retrieval subdomains on endosomes coalesce upon branched actin inhibition. (A-F) . HeLa cells were transfected with mCherry-RAB5 Q79L and were co-incubated with EGF-488 and anti-CD59 antibody for 20 min. During the last 5 min of uptake, either ( A-C ) no inhibitor or ( D-F ) 300 µM CK-666 was added to the media. Pearson’s and Manders’ correlation coefficients for each representative image are listed. (G). ImageJ was used to calculate Pearson’s correlation coefficient for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test. (H, I) . ImageJ was used to calculate Manders’ correlation coefficients (M1 and M2) for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Transfection, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: A Novel Hollow Fiber Infection Model (HFIM) for Antiviral PK/PD studies of CMV infection

doi: 10.64898/2026.03.13.710048

Figure Lengend Snippet: Circle and square lines represent the Towne (lab-adapted) and the Merlin (wild-type) strain, respectively. ( A, B ) represent the genome copies (copies/mL) and virus titer (PFU/mL), over time. ( C, D ) represent the log 10 fold change relative to quantified copies/mL and PFU/mL at day of inoculation.

Article Snippet: Human cytomegalovirus (HCMV) Towne (ATCC ® VR-977 TM) and the Merlin strains were used for the infection model.

Techniques: Virus

Journal: bioRxiv

Article Title: A Novel Hollow Fiber Infection Model (HFIM) for Antiviral PK/PD studies of CMV infection

doi: 10.64898/2026.03.13.710048

Figure Lengend Snippet: Ganciclovir treatment was administered 2 mg/hour every 12 hours, 3 days post-inoculation. Circle and square lines represent the control (no drug treatment) and drug treatment group. (A-D) represent the log10-fold change of genome copies (copies/mL) and virus titers (PFU/mL) for Towne and Merlin, respectively, over 72 hours post-treatment initiation.

Article Snippet: Human cytomegalovirus (HCMV) Towne (ATCC ® VR-977 TM) and the Merlin strains were used for the infection model.

Techniques: Control, Virus

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Endothelial cell-derived exosomes influence vascular smooth muscle cell phenotype and calcification-related gene expression. HAVSMCs were incubated for 8 days with 10 µg/mL exosomes derived from endothelial cells (ECs) in ECM (control), TNFα, TGFβ, or varying concentrations of TMAO (1–100 μM). ( A – D ) qPCR analysis of osteogenic markers RUNX2 and OPN, confirming transcriptional reprogramming toward an osteoblast-like phenotype. ( E ) TNAP (Tissue Non-Specific Alkaline Phosphatase) involved in vascular calcification and osteogenic transformation of VSMCs. Data are presented as mean ± SD from four independent biological replicates. Statistical significance was determined by one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. SMCM control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Derivative Assay, Gene Expression, Incubation, Control, Transformation Assay

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Differential effects of endothelial cell-derived exosomes on calcification of HAVSMCs, assessed by Alizarin Red staining. ( A – G ) Representative images of Alizarin Red staining in HAVSMCs after 8 days of culture with 10 µg/mL endothelial cell-derived exosomes (EC-EXOs) obtained from endothelial cell maintenance medium (ECM EC EXO), TNFα-stimulated EC exosomes (TNFα EC EXO), TGFβ-stimulated EC exosomes (TGFβ EC EXO), TMAO-treated EC exosomes (1 µM, 10 µM, and 50 µM TMAO EC EXO), and control smooth muscle cell medium (SMCM). ( H ) Quantification of Alizarin Red stain intensity was normalized to total protein concentration. Data are presented as mean ± SD from four independent biological replicates. Statistical significance was determined by one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05 vs. SMCM control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Derivative Assay, Staining, Control, Protein Concentration

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: β-catenin inhibition attenuates endothelial exosome-induced β-catenin activation in HAVSMCs. ( A , C ) Representative Western blot images showing non-phosphorylated (active) β-catenin protein expression in human aortic vascular smooth muscle cells (HAVSMCs) treated with endothelial cell-derived exosomes (EC-EXOs) obtained from TNFα-, TGFβ-, or TMAO-stimulated endothelial cells, in the presence or absence of the β-catenin transcriptional inhibitor ICG-001 for 8 days. β-actin was used as a loading control. ( B , D ) Quantitative densitometric analysis demonstrates a significant increase in β-catenin protein levels following EC-EXO treatment, which was markedly reduced upon β-catenin inhibition with ICG-001. Protein expression levels were normalized to β-actin and expressed as fold change relative to vehicle-treated controls. Data are presented as mean ± standard deviation (SD) from three independent biological replicates. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test to assess differences between EC-EXO treatment groups and the effect of β-catenin inhibition. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Derivative Assay, Control, Standard Deviation

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: β-catenin inhibition suppresses endothelial exosome-induced osteogenic gene expression in HAVSMCs. ( A – E ) Quantitative real-time PCR analysis of osteogenic gene expression in HAVSMCs treated with endothelial cell-derived exosomes (EC-EXOs) from TNFα-, TGFβ-, or TMAO-stimulated endothelial cells, in the presence of the β-catenin inhibitor ICG-001. Relative mRNA expression levels of ( A ) SM22A, ( B ) αSMA, ( C ) RUNX2, ( D ) osteopontin (OPN), and ( E ) tissue-nonspecific alkaline phosphatase (TNAP) were normalized to housekeeping genes and expressed relative to vehicle-treated control cells (0.1% v / v DMSO). EC-EXO co-treatment with ICG-001 significantly attenuated the expression of RUNX2, OPN, and TNAP, indicating that β-catenin signaling is required for endothelial exosome-induced osteogenic reprogramming of HAVSMCs. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was assessed using one-way ANOVA, followed by post-hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. CTL vehicle.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Inhibition, Gene Expression, Real-time Polymerase Chain Reaction, Derivative Assay, Expressing, Control

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Uptake kinetics of MemBright-labeled endothelial cell-derived exosomes by HAVSMC. Representative confocal microscopy images showing the time-dependent uptake of MemBright-labeled endothelial cell-derived exosomes by human aortic vascular smooth muscle cells (HAVSMCs). ( A ) HAVSMCs treated with control endothelial cell-derived exosomes (CTL EC EXO). ( B ) HAVSMCs treated with exosomes derived from endothelial cells exposed to 50 µM TMAO (TMAO EC EXO). Exosomes were labeled with MemBright (green), and cell nuclei were counterstained with Hoechst (blue). Images were acquired immediately after exosome addition (T = 0 h) and after 1, 3, and 4 h of incubation. Merged images illustrate progressive internalization and intracellular accumulation of exosomes over time, with 20× objective. All images were captured using a Leica confocal laser scanning microscope under identical acquisition settings. Scale bar: 194 µm.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Labeling, Derivative Assay, Confocal Microscopy, Control, Incubation, Laser-Scanning Microscopy

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: miR-222-3p overexpression promotes osteogenic signaling in HAVSMCs through activation of β-catenin pathway. ( A ) Quantitative PCR analysis confirming successful transfection of HAVSMCs with miR-222-3p mimic compared with the results for scrambled mimic control. Relative miR-222-3p expression levels were normalized to miR5S and expressed as fold change. ( B – F ) Quantitative PCR analysis of gene expression levels of RUNX2, OPN and TNAP in HAVSMCs after miR-222-3p mimic transfection for 48 h. ( G ) Representative Western blot images showing β-catenin protein expression in HAVSMCs following transfection with scrambled mimic or miR-222-3p mimic. ( H ) Quantitative densitometric analysis of protein expression levels of β-catenin protein expression levels were normalized to housekeeping protein and expressed relative to scrambled control. Data are presented as mean ± SD from independent biological replicates. Statistical significance was determined using unpaired two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. scrambled mimic control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Over Expression, Activation Assay, Real-time Polymerase Chain Reaction, Transfection, Control, Expressing, Gene Expression, Western Blot, Two Tailed Test