Journal: BMC Oral Health

Article Title: A comparative study of the epithelial regeneration capacities of two biomaterials in vitro

doi: 10.1186/s12903-025-06026-x

Figure Lengend Snippet: Investigation of reepithelialization-related biomarkers expressed by oral epithelial cells grown on different materials. Gene expression levels of KRT14 ( A ), KRT10 ( B ), ITGB-1 ( C ), ICAM-1 ( D ), IL-8 ( E ), IL-1β ( F ) after 3, 7 and 14 days were measured by qPCR. The 2 -ΔΔCt method was used for data analysis with GAPDH as the normalization reference gene. The Y-axis illustrates the relative fold change in expression levels compared to the day-3 TCP control, where the n-fold expression is defined as 1. Results are presented as mean ± SD from five independent donors. Statistical differences between the 3 substrate groups at the same evaluation time points are denoted with * and **, indicating p < 0.05 and p < 0.01, respectively

Article Snippet: RT-qPCR was performed using the following TaqMan gene expression assays (Applied Biosystems, Foster City, USA): KRT10 (Hs00166289_m1), KRT14 (Hs00265033_m1), ITGB-1 (Hs01127536_m1), ICAM-1 (Hs00164932_m1), IL-1β (Hs01555410_m1), IL-8 (Hs00174103_m1) at following conditions: 10 min of initial heating at 95 °C followed by 50 cycles at 95 °C for 15 s and at 60 °C for 1 min using a QuantStudio 3 device (Applied Biosystems, Foster City, USA).

Techniques: Gene Expression, Expressing, Control

Journal: bioRxiv

Article Title: Disease-Linked Regulatory DNA Variants and Homeostatic Transcription Factors in Epidermis

doi: 10.1101/2024.11.07.622542

Figure Lengend Snippet: a, Experimental diagram for a CRISPR-flow screen of all human TFs in primary keratinocytes. b, Distribution of KRT10 staining intensity across the three sorting groups. c, Comparison of enrichments for cells grown under progenitor maintenance or differentiation conditions suggests TFs with differential roles across epidermal differentiation. d, Guides for progenitor maintenance TFs are enriched in KRT10-high sorted cells, while guides for pro-differentiation TFs are depleted. e, Guides for differentiation factors are enriched in KRT10-low cells, while progenitor maintenance factors guides are depleted. f, Correlation between the KRT10-low and high sorted populations reveals outlier effects for known factors IRF6, SNAI2 and SP1. g, The effect of knock-outs on growth as the enrichment of guides in progenitor cells vs plasmid DNA (pDNA), compared with the effect on differentiation, represented by the enrichment of guides in KRT10-high or depletion from KRT10-low cells, whichever absolute value was higher. The interpretation of each quadrant in TF effects is an inset. “Day 0” refers to cells that were not differentiated, and instead were grown for four days while the other cells differentiated. pDNA, plasmid DNA. h, Overview of significant TFs identified by CRISPR-flow.

Article Snippet: For staining, cells were washed with Perm/Wash buffer, then stained with 1 µL anti-KRT10, AF647-conjugated antibody (Novus Bio, NBP2-47825AF647) per 1 million cells for 20 minutes in the dark at room temperature.

Techniques: CRISPR, Staining, Comparison, Plasmid Preparation

Journal: bioRxiv

Article Title: Disease-Linked Regulatory DNA Variants and Homeostatic Transcription Factors in Epidermis

doi: 10.1101/2024.11.07.622542

Figure Lengend Snippet: a , The correlation of SSC-A and KRT10 staining in CRISPR-flow agrees with prior results that cells enlarge and become more granular across differentiation. b , The number of UMIs by sample in CRISPR-flow. c, Essential gene guides are depleted from cells. d , Guides targeting DepMap “selective” genes (e.g., genes that reduce growth) and keratinocyte-specific factors were depleted by growth and a subset of TFs were enriched by growth. e , Combining high/low enrichment statistics into a single pseudo-P value for an effect leads to a depletion of unexpressed genes (see methods). P values for enrichment or depletion from the KRT10 high or low samples were combined into a pseudo-P value and the effect of different cutoffs (top panel) was evaluated for the depletion of un-expressed genes (middle panel, fraction of the expected number of unexpressed genes included, bottom panel, significance of the depletion of unexpressed genes by Fisher’s exact test). f , Calculation of an FDR rate for pseudo-P cutoffs based on the depletion of un-expressed genes as a function of prior belief in the true positive (TP) number. g , Diagram of how effects on growth can result in effects on the distribution of guides between progenitor and differentiated cells. h , The number of UMIs per gene as a histogram for genes in the given categories. i , PCA of guide enrichments reveal a primary axis of promoting/repressing differentiation, and a secondary axis correlated with whether the TF is more essential under differentiation or progenitor growth conditions.

Article Snippet: For staining, cells were washed with Perm/Wash buffer, then stained with 1 µL anti-KRT10, AF647-conjugated antibody (Novus Bio, NBP2-47825AF647) per 1 million cells for 20 minutes in the dark at room temperature.

Techniques: Staining, CRISPR

Journal: bioRxiv

Article Title: Disease-Linked Regulatory DNA Variants and Homeostatic Transcription Factors in Epidermis

doi: 10.1101/2024.11.07.622542

Figure Lengend Snippet: a, Experimental diagram for the perturb-seq time points. b, Gene expression UMAPs of keratinocyte marker genes for the three time points. c, - d, Guide effects on differentiation in perturb-seq as determined by Mann-Whitney tests for altered pseudotime trajectory at day 3 and day 6. e, Correlation of differentiation effects between CRISPR-flow (as mean change to the KRT10 High/Low distribution) and perturb-seq (as changes to mean pseudotime), both as Z-scored effect sizes, identifies the most potent TFs as outliers and highlights potential novel TFs (green labels). f, Enrichment of cells with the given guide (row labels) at the indicated position in the pseudotime trajectory (x axis). g, The effect of TF knock-outs on RNA expression for abundant RNAs dynamically expressed across differentiation. Colors represent the Z score for expression of the indicated RNA (x-axis) in cells with the given target (y-axis), vs other targets. h, Guide enrichment in differentiated cells of the strongest TFs (by significance) in CRISPR-flow and perturb-seq. i, Correlation of broken motif enrichment in MPRA with combined CRISPR-flow/perturb-seq effects on differentiation highlight AP-1 (red dots) and SP1.

Article Snippet: For staining, cells were washed with Perm/Wash buffer, then stained with 1 µL anti-KRT10, AF647-conjugated antibody (Novus Bio, NBP2-47825AF647) per 1 million cells for 20 minutes in the dark at room temperature.

Techniques: Expressing, Marker, MANN-WHITNEY, CRISPR, RNA Expression

Journal: bioRxiv

Article Title: Disease-Linked Regulatory DNA Variants and Homeostatic Transcription Factors in Epidermis

doi: 10.1101/2024.11.07.622542

Figure Lengend Snippet: a , UMAP of Day 3 differentiated keratinocytes colored by pseudotime, scaled from 1 to 10, in order of increasing differentiation. b , Markers for early differentiation (KRT10), late differentiation (SBSN), and basal cells (ITGA6) across pseudotime bins 1 to 10. c , RNA velocity analysis of Day 6 differentiated keratinocytes shows an RNA-velocity derived trajectory matching the pseudotime curve. This UMAP differs due to it being calculated by a different program (scanpy) based on a smaller subset of cells. d , Transcription factors are successfully depleted in target cells. The pink distribution represents the distribution of Z scores for expression of the target TF RNA in cells with guides against the target TF. Only TFs in the top half of expression are included, due to the limited sensitivity of single-cell RNA-seq. e , Enrichment of the 108 homeostasis TFs for TF PPIs, followed by transcription factor networks f and co-expression g . h , PPI network of TFs using STRING reveals a large network of interactions. i , Expression of three keratinocyte inflammation factors in Day 3 UMAPs. j , Expression of pro-inflammatory IL1 family cytokines IL1A, IL1B and IL18 in Day 3 UMAPs. k , Expression of IL1 cytokines across differentiation either in cells with any guide (solid lines) or with ATF4 or HIF1A knock-outs (dashed lines). l , RNA velocity and expression analysis of basal marker ITGA6, differentiation marker KRTDAP, IL1 cytokines (IL1A/B, IL18) and the IL1 receptor IL1R2 in Day 6 differentiated keratinocytes. A positive (green) velocity value represents induction, and negative (red) repression. m , Correlation of IL1 cytokine regulation and differentiation effects for TF knock-outs reveals only a slight negative correlation. n , Inflammation effects for TFs controlling for effects on differentiation. o , TFs with significant effects on inflammation, FDR<0.2. p , ATF4 and TFAP2A bind proximal to IL1A/B. ATF4 binding is reduced 2-fold over differentiation, consistent with a loss of activation at the later time points.

Article Snippet: For staining, cells were washed with Perm/Wash buffer, then stained with 1 µL anti-KRT10, AF647-conjugated antibody (Novus Bio, NBP2-47825AF647) per 1 million cells for 20 minutes in the dark at room temperature.

Techniques: Derivative Assay, Expressing, RNA Sequencing Assay, Marker, Binding Assay, Activation Assay