Fig 1.
Articles using the scratch assay vary by cell type.
(A) Progression and enumeration of articles found during qualitative literature review. (B) Pie chart of the total articles uncovered separated by general wound healing (often EMT) type or EndoMT. (C) Pie chart breakdown of total cell types used in the identified literature. KC = keratinocyte; FB = fibroblasts. EMT does not definitionally occur in FB, which are already of the mesenchymal phenotype.
Table 1.
Effect of different stimuli on scratch assay in keratinocytes, fibroblasts, and epithelial cells.
Migration rate of scratch assay in response to cell signalers, natural products, metabolic mediators, immune mediators, drugs, and other stimuli.
Fig 2.
Caffeine, but not chondroitin, inhibit scratch repair in keloid and healthy volunteer fibroblasts.
(A) Wound closure over 18 hours for healthy volunteer fibroblast line (HV-FB) treated with indicated concentrations of chondroitin. (B-C) Wound closure (B) and representative image (C) for HaCaT keratinocytes at 20hours with treatment of 12mg/mL of chondroitin at 18 hours. (D) Wound closure over 18 hours for HV-FB treated with indicated concentrations of caffeine. (E-F) Wound closure over 22 hours and representative images at 22 hours (F) for HV-FB or a commercially available fibroblasts cell line from a patient with keloid scaring (KEL-FB). (G) Wound closure over 22 hours for HV-FB and KEL-FB treated with 1mg/mL caffeine. (H) Wound closure at 14 hours for HV-FB and KEL-FB treated with indicated concentrations of caffeine. Results are representative of three independent experiments and displayed as mean + SEM for triplicate wells. * = p<0.05; ** = p<0.01; *** = p <0.001; for statistical comparison of area under the curve versus HV-FB under diluent stimulation conditions as determined by ANOVA with Sidak adjustment. Masking on representative images of scratch assay performed by Scratch App (BioTek).
Fig 3.
Impact of caffeine on vimentin expression and cytokine production.
(A) Representative images from vimentin stain in healthy volunteer (HV-) or keloid-derived (KEL-) fibroblast cell lines (FB). (B) Mean fluorescence intensity (MFI) per cell for vimentin for HV-FB and KEL-FB treated with indicated doses of caffeine. (C-I) Supernatant accumulation of cytokines and chemokines for cells treated with caffeine. Transforming growth factor beta 1 (TGFβ1; C), TGFβ2 (D), TGFβ3 (E), interleukin (IL-) 6 (F), IL-8 (G), RANTES (H), and CXCL1/GROα (I) are shown. Results are representative of two independent experiments and displayed as mean + SEM for triplicate wells. * = p<0.05; *** = p <0.001; for statistical comparison of area under the curve versus HV-FB under diluent stimulation conditions as determined by ANOVA with Sidak adjustment.
Fig 4.
Caffeine impacts metabolic function.
Seahorse assay was performed on fibroblast cell lines (FB) from healthy volunteer (HV) or keloid scars (Kel). Results for extracellular acidification rate (ECAR; a measure of glycolysis; A), mitochondrial (mito) ATP production (B), basal oxygen consumption rate (OCR; C), spare respiratory capacity (SRC; D), and ratio of basal ECAR to OCR (E) are shown. (F) Wound closure at 12 hours for keloid FB or healthy FB with treatment with diluent, the glycolysis inhibitor 2DG, or the mitochondrial OxPhos inhibitor rotenone. (G) Seahorse results for non-mitochondrial OCR for cells treated with diluent or caffeine. (H-I) Supernatant accumulation of interleukin (IL-) 6 (H) and CXCL1 (I). Results are representative of two independent experiments and displayed as mean + SEM with dots indicating replicate wells. * = p<0.05; ** = p<0.01; *** = p <0.001; **** = p < 0.0001 as determined by ANOVA with Sidak adjustment.
Fig 5.
Allicin impacts scratch outcomes and metabolic function.
(A-B) Wound closure as 12 hours (A) and representative image (B) for keloid (KEL-)or health volunteer (HV-) fibroblast cell lines (FB) after treatment with indicated doses of allicin. (C-F) Seahorse assay results for extracellular acidification rate (ECAR; C), spare respiratory capacity (SRC; D), mitochondrial (mito) ATP production (E), basal oxygen consumption rate (OCR; F), non-mitochondrial OCR (G), and ratio of basal ECAR to OCR (H) are shown. (I-H) Mean fluorescence intensity (MFI) per cell (I) and representative images (H) for vimentin for HV-FB and KEL-FB treated with indicated doses of allicin. (K-L) Supernatant accumulation of interleukin (IL-) 6 (K) and CXCL1 (L). Results are representative of two independent experiments and displayed as mean + SEM (A-G, I-L) or SD (H) for triplicate wells. * = p<0.05; ** = p<0.01; *** = p <0.001; **** = p < 0.0001 as determined by ANOVA with Sidak adjustment compared with HV in under similar conditions unless indicated; red brackets indicate statistical assessment for keloid FB while black brackets represent statistical assessment for HC-FB. Masking on representative images of scratch assay performed by Scratch App (BioTek). Negative values on scratch healing indicate inhibition of scratch repair (healing times that were slower than diluent control).
Fig 6.
Shikonin impacts scratch outcomes without influencing metabolic function.
(A-F) Seahorse assay results for keloid (KEL-) or health volunteer (HV-) fibroblasts (FB) for extracellular acidification rate (ECAR; A), basal oxygen consumption rate (OCR; B), spare respiratory capacity (SRC; C), mitochondrial (mito) ATP production (D), non-mitochondrial ATP production (E), and ratio of basal ECAR to OCR (F) are shown. (G) Wound closure as 12 hours for HV and KEL-FB after treatment with shikonin (10μM). (H) Mean fluorescence intensity (MFI) per cell for vimentin for HV-FB and KEL-FB treated with shikonin (10μM). (I-J) Supernatant accumulation of interleukin (IL-) 6 (I) and CXCL1 (J). Results are representative of three independent experiments and displayed as mean + SEM (A-E, G-J) or SD (F) for triplicate wells. * = p<0.05; ** = p<0.01; *** = p <0.001; **** = p < 0.0001 as determined by ANOVA with Sidak adjustment compared with HV in under similar conditions unless indicated.
Table 2.
Summary of impacts of compounds on healthy volunteer and keloid fibroblasts.
Summary of impacts of the glycolysis inhibitor (2DG), the mitochondrial ATP inhibitor (rotenone), caffeine, allicin, and shikonin on scratch assay healing time, extracellular acidification rate (ECAR), mitochondrial ATP production (Mito-ATP), non-Mito-ATP, the proinflammatory interleukin (IL-)6, and the neutrophil chemokine CXCL1.