Journal: BMJ Open Ophthalmology

Article Title: Protective effects of blue light-blocking shades on phototoxicity in human ocular surface cells

doi: 10.1136/bmjophth-2018-000217

Figure Lengend Snippet: Representative fluorescent microscopic images of immunostained cells with anti-pan-keratin antibody and its isotype control antibody (mouse IgG1). (A, B) Cells from human corneal surface origin (peripheral portion) and normal human dermal fibroblasts immunostained with anti-pan-keratin antibody, respectively. (C, D) Cells from human corneal surface origin (peripheral portion) and normal human dermal fibroblasts immunostained with an isotype control antibody (mouse IgG1), respectively. Scale bar=100 µm.

Article Snippet: Normal human dermal fibroblasts (NHDF, DS Pharma Biomedical, Osaka, Japan) were used as a negative control.

Techniques: Control

Journal: bioRxiv

Article Title: Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34 + hematopoietic progenitor cells and humanized mice

doi: 10.1101/2024.05.24.595672

Figure Lengend Snippet: (A, E) A dual luciferase reporter containing the 3’UTR of Akt (A) or Pin1 (E) was cotransfected into HEK293T cells along with miRNA mimics. Luciferase expression was assessed 24hrs post-transfection. The relative expression is shown as a percentage of Neg. Error bars represent the standard deviation from three separate experiments (**p<0.005 by unpaired t-test). (B) HEK293T cells were co-transfected with Akt-GFP and miRNA mimics or siRNA. Lysates were harvested 24hr post-transfection and immunoblotted for GFP and GAPDH. Quantification from one representative blot shows relative expression levels of Akt-GFP compared to Neg (normalized to GAPDH). (C) Quantification of (B) from three separate experiments (*p<0.05, **p<0.005, ***p<0.0005 [one-way ANOVA with Tukey’s multiple comparison test]). (D) NHDF cells were transfected with miRNA mimics, negative control (Neg), or siRNA. RNA has harvested 72hr post-transfection, and quantitative RT-PCR for Akt was performed. Expression levels were normalized to 18S and compared to Neg (*p<0.05, **p<0.005, ***p<0.0005 [one-way ANOVA with Tukey’s multiple comparison test]). (F) NHDF were transfected with miRNA mimics or siRNA. Lysates were harvested 72hr post-transfection and immunoblotted for Pin1, Akt, and GAPDH. Quantification from one representative blot shows relative expression levels of Akt compared to Neg (normalized to GAPDH). (G) Quantification of (F) from three separate experiments (*p<0.05, ***p<0.0005 [one-way ANOVA with Tukey’s multiple comparison test]).

Article Snippet: HEK293 and adult normal human dermal fibroblasts (NHDF) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone), 100 units/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml glutamine (Thermofisher).

Techniques: Luciferase, Expressing, Transfection, Standard Deviation, Comparison, Negative Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34 + hematopoietic progenitor cells and humanized mice

doi: 10.1101/2024.05.24.595672

Figure Lengend Snippet: (A,B) NHDF were infected with WT, ΔmiR-UL36/112/148D, or Mock infected for 48hr, serum starved overnight, and then stimulated +/−EGF for 15 minutes. Lysates were harvested and immunoblotted for Akt phosphorylated at T308 (A) or S473 (B) as well as total Akt, HCMV IE2, and GAPDH. Quantification from one representative blot shows relative expression levels of p-Akt compared to Neg (normalized to GAPDH). (C, D) Quantification of p-Akt levels from (A, B), respectively, from three separate experiments (comparing +EGF conditions *p<0.05, ***p<0.0005, ****p<0.0001 [two-way ANOVA with Tukey’s multiple comparison test]). (E, F) Ratio of p-Akt to total Akt levels in WT and ΔmiR-UL36/112/148D from (A, B), respectively, from three separate experiments, normalized to WT (*p<0.05 [unpaired t-test]).

Article Snippet: HEK293 and adult normal human dermal fibroblasts (NHDF) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone), 100 units/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml glutamine (Thermofisher).

Techniques: Infection, Expressing, Comparison

Journal: bioRxiv

Article Title: Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34 + hematopoietic progenitor cells and humanized mice

doi: 10.1101/2024.05.24.595672

Figure Lengend Snippet: (A, B, E, F) NHDF were infected with WT, ΔmiR-UL36/112/148D, or Mock infected for 48hr, serum starved overnight, and then stimulated +/−EGF for 15 minutes. Lysates were harvested and immunoblotted for indicated phosphorylated and total proteins as well as HCMV IE2 and GAPDH. Quantification from one representative blot shows relative expression levels of p-protein compared to Neg (normalized to GAPDH). (C, D, G, H) Quantification of (A, B, E, F), respectively, from three separate experiments (comparing +EGF conditions, **p<0.005, ***p<0.0005, ****p<0.0001 [two-way ANOVA with Tukey’s multiple comparison test]).

Article Snippet: HEK293 and adult normal human dermal fibroblasts (NHDF) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone), 100 units/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml glutamine (Thermofisher).

Techniques: Infection, Expressing, Comparison

Journal: bioRxiv

Article Title: Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34 + hematopoietic progenitor cells and humanized mice

doi: 10.1101/2024.05.24.595672

Figure Lengend Snippet: (A, B) NHDF were plated on coverslips and infected with WT, ΔmiR-UL36/112/148D, or Mock infected. Cells were fixed 72 hpi and stained for actin (phalloidin), FOXO3a, and nuclei (DAPI). (A) Representative images are shown. White dotted line shows outline of the nucleus in FOXO3a images. Scale bar, 20μM. (B) Image J software was used to quantify the average intensity of FOXO3a in the nucleus and the entire cell and graphed as a percentage of nuclear FOXO3a levels normalized to the whole cell. Error bars represent the standard error of the mean for 38-44 cells from each condition from three separate experiments (****p<0.0001 [one-way ANOVA with Tukey’s multiple comparison test]). (C-E) NHDF were infected with WT, ΔmiR-UL36/112/148D, or Mock infected for 72hr and RNA has harvested. Quantitative RT-PCR was performed using specific primers for MEIP (C), IP1 (D), or IP2 (E). Expression levels were normalized to GAPDH and compared to Mock (**p<0.003, ***p<0.0007, ****p<0.0001 [one-way ANOVA with Tukey’s multiple comparison test]).

Article Snippet: HEK293 and adult normal human dermal fibroblasts (NHDF) were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone), 100 units/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml glutamine (Thermofisher).

Techniques: Infection, Staining, Software, Comparison, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Functionalization of Bacterial Cellulose with the Antimicrobial Peptide KR-12 via Chimerical Cellulose-Binding Peptides

doi: 10.3390/ijms25031462

Figure Lengend Snippet: Solution peptide effect on NHDF viability assessed through metabolic activity. All data are normalized to the metabolic activity of untreated control cells indicated by the red dashed line; 70% activity is indicated by the grey dashed line. Data are presented as the mean and standard deviation from three independent experiments ( n = 3 per experiment). Statistical differences determined by an ANOVA followed by the Tukey’s HSD test (* p < 0.05, ** p < 0.01, *** p < 0.005).

Article Snippet: Komagataeibacter hansenii NQ5 ( K. hansenii ATCC 53582), normal human dermal fibroblasts (NHDFs; American Type Culture Collection: CRL-2565), Staphylococcus aureus (ATCC 43866, strain designation: LRA 44.01.83), Escherichia coli (ATCC 33694, strain designation: HB101) and Pseudomonas aeruginosa (ATCC 29260, strain designation: PA-103) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA, while human endothelial keratinocyte cells (HaCaT, catalog number: T0020001) were purchased from AddexBio, San Diego, CA, USA.

Techniques: Activity Assay, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Functionalization of Bacterial Cellulose with the Antimicrobial Peptide KR-12 via Chimerical Cellulose-Binding Peptides

doi: 10.3390/ijms25031462

Figure Lengend Snippet: Functionalized BC effect on NHDF and HaCaT viability. Normalized metabolic activity of ( A , B ) NHDF and ( C , D ) HaCaT after exposure to unfunctionalized BC (BC control) and BC functionalized with Long-CBP-KR12 and Short-CBP-KR12 normalized to untreated cells. Both the ( A , C ) opaque and ( B , D ) transparent BC were evaluated. All data are normalized to the metabolic activity of untreated control wells indicated by the red dashed line, and 70% activity is indicated by the grey dashed line. Data are presented as the mean and standard deviation from three independent experiments ( n = 3 per experiment). No statistical differences determined by ANOVA.

Article Snippet: Komagataeibacter hansenii NQ5 ( K. hansenii ATCC 53582), normal human dermal fibroblasts (NHDFs; American Type Culture Collection: CRL-2565), Staphylococcus aureus (ATCC 43866, strain designation: LRA 44.01.83), Escherichia coli (ATCC 33694, strain designation: HB101) and Pseudomonas aeruginosa (ATCC 29260, strain designation: PA-103) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA, while human endothelial keratinocyte cells (HaCaT, catalog number: T0020001) were purchased from AddexBio, San Diego, CA, USA.

Techniques: Activity Assay, Control, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Resveratrol-Enriched Rice Attenuates UVB-ROS-Induced Skin Aging via Downregulation of Inflammatory Cascades

doi: 10.1155/2017/8379539

Figure Lengend Snippet: The levels of ROS in NHDFs treated as indicated for 24 h were measured using flow cytometry with DCFH-DA dye. The number of cells is plotted against the dichlorofluorescein fluorescence detected by the FL-1 channel (a). The relative ROS production of the cells is shown in each histogram (b). Values are mean ± SEM. The labels # and ∗ indicate significant differences ( p < 0.05) when compared with the normal control and UV (+) control, respectively. ### p < 0.001 versus the normal control, ∗∗ p < 0.01 versus the UVB-irradiated control. NC is normal control, Ctl is UVB control, NR is normal rice, RR is resveratrol-enriched rice, and R is resveratrol. NR and RR were treated in μ g/mL, and R was treated in μ M.

Article Snippet: Normal human dermal fibroblast cells (NHDFs) were obtained by skin biopsy from a healthy young male donor (MCTT Core Inc., Seoul, Korea).

Techniques: Flow Cytometry, Fluorescence, Control, Irradiation

Journal: Annals of the rheumatic diseases

Article Title: RUNX1 is expressed in a subpopulation of dermal fibroblasts and is associated with disease severity of systemic sclerosis

doi: 10.1016/j.ard.2025.10.033

Figure Lengend Snippet: TGF- β 1 increases the RUNX1 expression in SSc fibroblasts and inhibition of RUNX1 reduces ECM markers. (A) Western blot of 3 isolated fibroblasts lines treated with TGF- β 1. The blot shows all isoforms of RUNX1a, b, and c that are overexpressed under the TGF- β 1 stimulation. (B) Schematic graph illustrating the timeline for the culture and TGF- β 1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and normal human dermal (NHD) fibroblast cells. RUNX1 expression rate in samples treated with TGF- β 1 (in red) vs control (in blue) for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the 2 SSc-isolated fibroblast lines at 12 hours after exposure vs the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF- β 1 treatment vs the baseline. Data from B to D were obtained through publicly available data of GSE12493 . (E) Schematic graph showing 2 lines of SSc-isolated fibroblasts treated with siRNA against RUNX1 (siRUNX1) and nontargeting control siRNA (siNC). (F) UMAP projection and dot plot of RUNX1 and CBFB of the single-cell RNA-seq data. (G) UMAP of 10 fibroblast clusters (0–9) for siR-UNX1 and siNC. (H) Cell proportion of siRUNX1 and siNC per cluster. (I) Top 4 upregulated and downregulated marker genes per cluster. (J) Top 15 enriched pathways that are significantly represented across siRUNX1 and siNC (K) Bar plots showing the percentage of cells expressing COL1A1, FN1, COL4A1, LUM, ACTA2, LGR5, COL8A1, COMP , and THBS1 per condition (red: siRUNX1, green: siNC) or per cluster. (L) Module score for extracellular matrix organisation pathway per cluster and per condition. (M) Feature plot of the ECM module score. dcSSc, diffuse cutaneous SSc; DEG, differentially expressed gene; ECM, extracellular matrix; RUNX1, runt-related transcription factor 1; siRUNX1, siRNA targeting RUNX1; SSc, systemic sclerosis; TGF- β , transforming growth factor- β ; UMAP, uniform manifold approximation and projection.

Article Snippet: We then analysed a previously generated DNA microarray dataset (National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO): GSE12493 ) consisting of 2 independent SSc fibroblast cell lines, 1 healthy control fibroblast cell line (isolated in parallel), and 1 normal human dermal (NHD) fibroblast cell line obtained from American Type Culture Collection (ATCC), treated with 50 pM TGF- β 1 [ ] ( ).

Techniques: Expressing, Inhibition, Western Blot, Isolation, Control, Single Cell, RNA Sequencing, Marker

Journal: Annals of the rheumatic diseases

Article Title: RUNX1 is expressed in a subpopulation of dermal fibroblasts and is associated with disease severity of systemic sclerosis

doi: 10.1016/j.ard.2025.10.033

Figure Lengend Snippet: RUNX1 contributes to fibroblast activation, proliferation and contraction. (A) RUNX1 western blot of CRISPR-generated RUNX1 KO and wild-type (WT) fibroblasts under the TGF- β 1 stimulation vs control. RUNX1 isoforms of a, b, and c were marked in the blot by arrows. (B) α -SMA and RUNX1 IF staining of KO and WT fibroblasts under the TGF- β 1 stimulation vs control. (C) α -SMA western blot of KO and WT fibroblasts under the TGF- β 1 stimulation vs control. (D) ACTA2 mRNA expression of KO and WT fibroblasts under the TGF- β 1 induction vs control. (E) Fold change expression of FN1, COL1A1, LUM , and SFRP4 in TGF- β 1-induced SSc fibroblasts treated with Ro5–3335 compared to control (3 lines of SSc fibroblasts, 2 replicates each). (F) Proliferation curve of normal human dermal (NHD) fibroblasts in the presence and absence of Ro5–3335. (G,H) The 3D collagen contraction assays, fixed (G) and floating (H) models, of NHD fibroblasts treated with Ro5–3335 (4 replicates for each condition). SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. Negative control is collagen matrix with no fibroblasts. The overhead pictures represent 1 replicate for each condition. (I) 3D self-assembled (SA) tissue constructs from the healthy- and SSc-isolated fibroblast lines with donors’ clinical characteristics. H&E staining of representative untreated and Ro5–3335-treated tissues. (J) Tissue area fold change of each cell line over the control for healthy and SSc SA tissues. Data from 3 healthy and 4 SSc lines, 3 replicates per line, repeated in 2 independent sets. (K) Change in area of an SSc-isolated SA tissue when treated for 1, 2, or 3 weeks with Ro5–3335 compared to control (Student’s t test P value: **.001-.01, ****<.0001 in GraphPad Prism v9). α -SMA, alpha smooth muscle actin; H&E, haematoxylin and eosin; KO, knockout; RUNX1, runt-related transcription factor 1; SSc, systemic sclerosis; TGF- β , transforming growth factor- β ;Clustered Regularly Interspaced Palindromic Repeats (CRISPR),Smad Family Member 3 (SMAD3),Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Not Applicable (N/A), Quantitative Polymerase Chain Reaction (QPCR), Quantitative Polymerase Chain Reaction, Immunofluorescenc (IF).

Article Snippet: We then analysed a previously generated DNA microarray dataset (National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO): GSE12493 ) consisting of 2 independent SSc fibroblast cell lines, 1 healthy control fibroblast cell line (isolated in parallel), and 1 normal human dermal (NHD) fibroblast cell line obtained from American Type Culture Collection (ATCC), treated with 50 pM TGF- β 1 [ ] ( ).

Techniques: Activation Assay, Western Blot, CRISPR, Generated, Control, Staining, Expressing, Positive Control, Negative Control, Construct, Isolation, Knock-Out, Real-time Polymerase Chain Reaction