Journal: JID Innovations

Article Title: Identification of Collagen-Suppressive Agents in Keloidal Fibroblasts Using a High-Content, Phenotype-Based Drug Screen

doi: 10.1016/j.xjidi.2023.100248

Figure Lengend Snippet: Study schema. Primary human fibroblast and human keloid fibroblast lines were plated into 96-well plates 1 day before drug treatment. One hundred ninety-nine kinase inhibitors from the HMS LINCS collection were added to replicate 96-well plates. One plate each was placed in 20% oxygen or 1% oxygen incubators concurrently. Twenty-four hours after drug addition, the supernatants were removed and assayed for Col1a1 by anti-Col1a1 ELISA, whereas Cell-TiterGo was used to determine viability. The percentage CI for each drug is calculated as the drug COL1A1 levels relative to the average COL1A1 levels from multiple DMSO wells in that specific drug plate (ie, Drug col1a1 ÷ DMSO average col1a1 ) × 100. The percentage viability is defined as the drug viability levels to the average viability from multiple DMSO wells in that specific drug plate (ie, Drug viability ÷ DMSO average viability ) × 100. The CI norm is an approximation of the amount of collagen suppression per cell (ie, divided by viability): (drug col1a1 ÷ DMSO average col1a1 )/(drug viability ÷ DMSO average viability ). The mean normalized CI index (denoted as CI ¯ norm ) is the average of the CI norm from all the cell lines. The most suppressive agent (CGP60474) was then subjected to secondary validation with confocal, dose–response curves, phosphokinome, and western blot analyses. O 2 denotes oxygen. CI, collagen inhibition; CI norm , normalized collagen inhibition index; HMS, Havard Medical School.

Article Snippet: After 24 hours of drug treatment, the supernatant was removed and saved at −80 o C. Collagen I secretion (Col1a1) and IL-6 secretion were measured using ELISA after optimization by Human Procollagen I alpha 1/Col1a1 PicoKine ELISA Kit and Human IL-6/Interleukin-6 ELISA Kit PicoKine from Boster Bio (Pleasanton, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Western Blot, Inhibition

Journal: JID Innovations

Article Title: Identification of Collagen-Suppressive Agents in Keloidal Fibroblasts Using a High-Content, Phenotype-Based Drug Screen

doi: 10.1016/j.xjidi.2023.100248

Figure Lengend Snippet: Screen of 199 kinase inhibitors on normal and KFs. ( a ) BJ fibroblasts and 4 KFs were treated with kinase inhibitor drug panel at 1% oxygen. Relative viability was measured with CellTiter-Glo and normalized to DMSO. Extracellular COL1A1 was measured using ELISA and normalized to DMSO. Each blue dot represents the percentage viability (drug viability ÷ DMSO average viability [Y-axis]) graphed against the percentage collagen inhibition (drug col1a1 ÷ DMSO average col1a1 [X-axis]) for a single drug. The experiment was replicated 5 times each using a different cell line once. ( b ) The mean CI norm ( CI ¯ norm ; ie, the average CI norm for BJ, KF1, KF4, KF5, and KF6; orange line) is ranked from most suppressive (left side) to most inductive (right side). The SDs for each drug across the 5 biological replicates (ie, the 5 cell lines) are shown as blue bars. ( c ) Table showing the top 10 most COL1A1-suppressive and collagen-inducive drugs ranked by CI ¯ norm . Akt, protein kinase B; KF, keloid fibroblast; MET, MAPK/extracellular signal–regulated kinase; PI3K, phosphoinositide 3-kinase.

Article Snippet: After 24 hours of drug treatment, the supernatant was removed and saved at −80 o C. Collagen I secretion (Col1a1) and IL-6 secretion were measured using ELISA after optimization by Human Procollagen I alpha 1/Col1a1 PicoKine ELISA Kit and Human IL-6/Interleukin-6 ELISA Kit PicoKine from Boster Bio (Pleasanton, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition

Journal: JID Innovations

Article Title: Identification of Collagen-Suppressive Agents in Keloidal Fibroblasts Using a High-Content, Phenotype-Based Drug Screen

doi: 10.1016/j.xjidi.2023.100248

Figure Lengend Snippet: Cellular screen for CGP60474. ( a ) Confocal microscopy shows loss of intracellular COL1A1 (green) in the BJ fibroblast and 5 KF lines after exposure to 1 μM CGP60474 for 24 hrs. DAPI stain is shown in blue. Bar = 20 μm ( b ) Dose–response curves for CGP60474 in 2 KF lines showing normalized (to DMSO) viability and extracellular COL1A1. ( c ) Effect of 1 μM CGP60474 on both Col1a1 and Col7a1 in KF-1762, KF1, KF4, and KF6 showing suppression of both collagens. hr, hour; KF, keloid fibroblast.

Article Snippet: After 24 hours of drug treatment, the supernatant was removed and saved at −80 o C. Collagen I secretion (Col1a1) and IL-6 secretion were measured using ELISA after optimization by Human Procollagen I alpha 1/Col1a1 PicoKine ELISA Kit and Human IL-6/Interleukin-6 ELISA Kit PicoKine from Boster Bio (Pleasanton, CA).

Techniques: Confocal Microscopy, Staining

Journal: JID Innovations

Article Title: Identification of Collagen-Suppressive Agents in Keloidal Fibroblasts Using a High-Content, Phenotype-Based Drug Screen

doi: 10.1016/j.xjidi.2023.100248

Figure Lengend Snippet: CGP60474 and MAPK signaling. ( a ) Representative phosphokinome array for KF-1762 (Proteome Profiler—Human Phospho-Kinase Array—(R&D Systems, ARY003C) showing most upregulated phosphoproteins (p-Erk T202/Y204,T185/Y187 , p-Hsp27 S78/S82 , p-Gsk-3α/β S21/S9 ). ( b ) Normalized (to background) and relative (to DMSO) densitometry units for the p-Erk T202/Y204,T185/Y187 , p-Hsp27 S78/S82 , and p-Gsk-3α/β S21/S9 based on 3 independent phosphokinome arrays. ( c ) Western blots showing levels of COL1A1, p-ERK, total ERK, and GAPDH in KF-1762 and KF5 at 20 and 1% oxygen. ERK, extracellular signal–regulated kinase; KF, keloid fibroblast; p-ERK, phosphorylated extracellular signal–regulated kinase; p-Gsk, phosphorylated Gsk; p-Hsp27, phosphorylated Hsp27.

Article Snippet: After 24 hours of drug treatment, the supernatant was removed and saved at −80 o C. Collagen I secretion (Col1a1) and IL-6 secretion were measured using ELISA after optimization by Human Procollagen I alpha 1/Col1a1 PicoKine ELISA Kit and Human IL-6/Interleukin-6 ELISA Kit PicoKine from Boster Bio (Pleasanton, CA).

Techniques: Western Blot

Journal: JID Innovations

Article Title: Identification of Collagen-Suppressive Agents in Keloidal Fibroblasts Using a High-Content, Phenotype-Based Drug Screen

doi: 10.1016/j.xjidi.2023.100248

Figure Lengend Snippet: CGP60474 and IL-6. ( a ) Various primary KFs (different color circles) were treated with 1 and 3 μM CGP60474 for 24 hrs in either 20 oxygen or 1% oxygen (raw data are in Supplementary Table S5 ). Levels of secreted IL-6 (pg/ml) were assayed by ELISA (human IL-6/IL-6 ELISA Kit PicoKine). Except for KF4, in general, there were decreases in secreted IL-6. ( b ) Effect of adding IL-6 (100 nM) and anti–IL-6R (3 μg) on COL1A1, p-ERK, and total ERK in KF6. Erk, extracellular signal–regulated kinase; hr, hour; IL-6R, IL-6 receptor; KF, keloid fibroblast; p-Erk, phosphorylated extracellular signal–regulated kinase; RLU, Relative Luminescence Units.

Article Snippet: After 24 hours of drug treatment, the supernatant was removed and saved at −80 o C. Collagen I secretion (Col1a1) and IL-6 secretion were measured using ELISA after optimization by Human Procollagen I alpha 1/Col1a1 PicoKine ELISA Kit and Human IL-6/Interleukin-6 ELISA Kit PicoKine from Boster Bio (Pleasanton, CA).

Techniques: Enzyme-linked Immunosorbent Assay