Journal: The Journal of Clinical Investigation

Article Title: Staphylococcus aureus accessory gene regulator quorum-sensing system inhibits keratinocyte lipid enzymes and delays wound repair

doi: 10.1172/JCI190411

Figure Lengend Snippet: ( A ) Representative images of murine wounds 7 days after infection with indicated S . aureus mutants. ( B ) Bacteria recovered from day 7 murine wounds infected with S . aureus mutants. ( C ) Quantification of closure on day 7 of murine wounds infected with S . aureus mutants. ( D ) LDH release from scratch-wounded primary NHEKs 24 hours after treatment with CM of S . aureus mutants (3 × 10 7 CFU/mL equivalent). ( E ) Scratch-wounded NHEK migration after 24-hour treatment with CM of S . aureus mutants (3 × 10 7 CFU/mL equivalent). ( F ) Proliferation measured with MTT assay of NHEKs after 24-hour treatment with CM of S . aureus mutants (3 × 10 7 CFU/mL equivalent). ( G ) KI67 gene expression in proliferating NHEKs treated for 24 hours with CM of S . aureus mutants (3 × 10 7 CFU/mL equivalent). One-way ANOVA followed by Bonferroni’s multiple-comparison adjustment for more than 2 groups ( B – G ). Experiments were performed at least twice. Data represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Primary NHEKs (ATCC, PCS-200-010) were cultured in EpiLife medium containing 60 μM CaCl 2 (Gibco, MEPI500CA) supplemented with Human Keratinocyte Growth Supplement (Gibco, S0015) and antibiotic-antimycotic (penicillin 100 U/mL, streptomycin 100 U/mL, and amphotericin B 250 ng/mL; Gibco, 15240062) at 37°C, 5% CO 2 .

Techniques: Infection, Bacteria, Migration, MTT Assay, Gene Expression, Comparison

Journal: Nature Communications

Article Title: Identification of functional non-coding variants associated with orofacial cleft

doi: 10.1038/s41467-025-61734-w

Figure Lengend Snippet: a – c UCSC browser views of the human genome, GRCh37/hg19, focused on the OFC-associated SNPs at a , b IRF6 and c FOXE1 . OFC SNPs, SNPs identified by GWAS and evaluated by MPRA at each locus. One SNP at the FOXE1 locus, which was tested in the MPRA but lacked significant effects in it, rs1877431, is outside of the range presented here. MPRA-sig SNPs, SNPs with allele-specific effects in the MPRA. IRF6 enhancers, FOXE1 enhancers, and enhancers for the indicated gene identified using the activity-by-contact (ABC) method and datasets from NHEK (see Methods). Gray arrows, GWAS-meta-analysis P values of the indicated SNPs. Two-sided P values are calculated with an inverse-variance weighted fixed-effects meta-analysis of a transmission disequilibrium test without adjustment for multiple testing and a logistic regression (that had 18 principal components of ancestry as covariates). GM12878 B-cell derived cell line, H1-ESC embryonic stem cells, K562 myelogenous leukemia cell line, HepG2 liver cancer cell line, HUVEC human umbilical vein endothelial cells, HMEC human mammary epithelial cells, HSMM human skeletal muscle myoblasts, NHEK normal human epidermal keratinocytes, NHLF normal human lung fibroblasts. CS 13-20, Carnegie stages for human embryonic face explants . Colored bars, inferred chromatin state from combinatorial analysis of multiple chromatin mark datasets . Orange and yellow, active and weak enhancer element, respectively; bright red, active promoter; light red, weak promoter; purple, inactive/poised promoter; blue, insulator; light green, weakly transcribed; gray, Polycomb repressed; light gray, heterochromatin/repetitive. Additional color bars in the facial explants dataset, green, transcription; green-yellow, transcription weak enhancer; purple, bivalent promoter; light purple, poised promoter.

Article Snippet: Human Epidermal Keratinocytes from neonatal foreskin (HEKn; primary neonatal keratinocytes) purchased from ATCC (ATCC Catalog no. PCS-200-010) were maintained and cultured as per the manufacturer’s instructions in dermal cell basal medium (ATCC Catalog no. PCS-200-030) supplemented with keratinocyte growth kit (ATCC, Catalog no. PCS-200-040).

Techniques: Activity Assay, Transmission Assay, Derivative Assay

Journal: Nature Communications

Article Title: Identification of functional non-coding variants associated with orofacial cleft

doi: 10.1038/s41467-025-61734-w

Figure Lengend Snippet: a Bar chart of MPRA and luciferase reporter activities for the indicated SNPs at various loci in GMSM-K using elements of the indicated length centered on the SNP. MPRA data are represented as mean ± standard error of the mean (SEM) from the indicated number of replicates. Statistical significance ( P value, two-tailed) of the difference between major and minor allele’s reporter activity is determined by Welch’s t -test, followed by Benjamini–Hochberg false discovery rate correction. P (MPRA) = 0.0452 (rs11110348), 0.0057 (rs661849), 0.9097 (rs642961), 0.6942 (rs4844939), 0.4487 (rs12104876), 0.0238 (rs75436877), 0.7100 (rs79482068), 0.0734 (rs79792381), 0.0125 (rs201265), 0.1206 (rs10124184), 0.0067 (rs10984103), 0.0458 (rs4812449), 0.2945 (rs57369620). For luciferase reporter activities, data are represented as mean ± standard deviation (SD) from four independent experiments. Statistical significance ( P value, two-tailed) is determined by Student’s t -test. P (luciferase) = 0.0006 (rs11110348), <0.0001 (rs661849), 0.3822 (rs642961), <0.0001 (rs4844939), 0.14 (rs12104876), <0.0001 (rs75436877), 0.0693 (rs79482068), 0.5115 (rs79792381), 0.002 (rs201265), <0.0001 (rs10124184), <0.0001 (rs10984103), <0.0001 (rs4812449), 0.1421 (rs57369620). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS non-significant. b – d Scattered dot plots of relative luciferase activity (using the longer elements described in Results) for non-risk and risk alleles of rs11119348, rs661849 and rs10984103 in primary neonatal keratinocytes (HEKn). Value of 1 is that of the empty pGL3 promoter vector. Data are represented as mean ± standard deviation (SD) from four independent experiments. Statistical significance ( P value, two-tailed) is determined by Student’s t -test and P value is indicated on the plot. e – g Scattered dot plot of relative levels of e , f IRF6 or g FOXE1 mRNA in edited induced oral epithelium (iOE) cells homozygous for the non-risk or risk alleles of each SNP, as indicated, assessed by qRT-PCR. Expression levels are normalized against those of ACTB, GAPDH, HPRT1, UBC and CDH1 . Data were represented as mean ± SD from e , f six replicates or g nine replicates of cells harboring each genotype, as indicated in the plot. Each dot represents three technical qPCR replicates. Statistical significance ( P value, two-tailed) is determined by Student’s t -test and P value is indicated on the plot. Source data are provided as a Source Data file.

Article Snippet: Human Epidermal Keratinocytes from neonatal foreskin (HEKn; primary neonatal keratinocytes) purchased from ATCC (ATCC Catalog no. PCS-200-010) were maintained and cultured as per the manufacturer’s instructions in dermal cell basal medium (ATCC Catalog no. PCS-200-030) supplemented with keratinocyte growth kit (ATCC, Catalog no. PCS-200-040).

Techniques: Luciferase, Two Tailed Test, Activity Assay, Standard Deviation, Plasmid Preparation, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Identification of functional non-coding variants associated with orofacial cleft

doi: 10.1038/s41467-025-61734-w

Figure Lengend Snippet: a Consensus ETS2 binding motif from the JASPAR database of transcription factor DNA-binding preferences (Matrix ID: MA1484.1) and alignment of the variant site in several mammals. The risk allele (C) is the reference allele but has a lower frequency than the non-risk allele (T) in most populations. The risk allele improves the match to the ETS2 binding site which remains partial. b , c Percent input identified by ChIP-qPCR for anti-ETS2 and anti-H3K27Ac respectively in iOE cells heterozygous for rs661849 using primers specific to the IRF6 −22 kb enhancer site or, as a negative control, to a region 103.7 kb upstream IRF6 transcription start site lacking ATAC-Seq and H3K27Ac ChIP-Seq signals in HIOEC or NHEK. Error bars refer to three ChIP replicates and expressed as mean ± SD. Each dot represents three technical qPCR replicates. Statistical significance ( P value, two-tailed) is determined by Student’s t -test and P value is indicated on the plot. NS non-significant. d Sequencing of anti-ETS2 and anti-H3K27Ac ChIP-PCR product of cells heterozygous for rs661849 using the indicated antibody. e Scattered dot plot of relative luciferase activity of the IRF6 −22 kb reporter construct (longest version) harboring the risk allele of rs661849 in control versus ETS2 -depleted primary neonatal keratinocytes (HEKn). Data are represented as mean ± SD from three independent experiments. Statistical significance ( P value, two-tailed) is determined by Student’s t -test and P value is indicated on the plot. f Scattered dot plot of relative levels of IRF6 mRNA in control versus ETS2 -depleted HEKn assessed by qRT-PCR. Expression levels of IRF6 are normalized against ACTB . Data are represented as mean ± SD from three replicates. Each dot represents three technical qPCR replicates. Statistical significance ( P value, two-tailed) is determined by Student’s t -test and P value is indicated on the plot. g Model showing binding of ETS2 to the IRF6 −22 kb enhancer, which is favored by the risk allele and which reduces IRF6 expression. Source data are provided as a Source Data file.

Article Snippet: Human Epidermal Keratinocytes from neonatal foreskin (HEKn; primary neonatal keratinocytes) purchased from ATCC (ATCC Catalog no. PCS-200-010) were maintained and cultured as per the manufacturer’s instructions in dermal cell basal medium (ATCC Catalog no. PCS-200-030) supplemented with keratinocyte growth kit (ATCC, Catalog no. PCS-200-040).

Techniques: Binding Assay, Variant Assay, ChIP-qPCR, Negative Control, ChIP-sequencing, Two Tailed Test, Sequencing, Luciferase, Activity Assay, Construct, Control, Quantitative RT-PCR, Expressing