human primary kcs  (AcceGen Biotechnology)

Journal: Skin Health and Disease

Article Title: Anti‐inflammatory control of human skin keratinocytes by targeting nuclear transport checkpoint

doi: 10.1002/ski2.356

Figure Lengend Snippet: NTCI treatment of the human primary KCs inhibits proinflammatory signalling to the nucleus through reducing the nuclear translocation of NF‐κB RelA and STAT3. The human primary KCs were pretreated with 10 μM NTCI (cSN50.1 peptide) 30 min before stimulation with a mix of two cytokines: TNF‐α (10 ng/ml) and IL‐17A (20 μg/ml) ² and incubated overnight. The nuclear content of the two SRTFs, NF‐κB RelA and pSTAT3 (phosphorylated STAT3), was determined by a quantitative immunoblotting analysis using the LI‐COR Odyssey Infrared Imaging System. The data is presented as a mean + S.E.M. ( n = 4). The statistical significance of a difference between the nuclear content of two SRTFs in the TNF‐α/IL‐17A‐stimulated cells (Stimulated) and stimulated cells treated with the NTCI, cSN50.1 peptide (Stim. + cSN50.1) was determined by an ordinary one‐way ANOVA with uncorrected Fisher’s LSD test for multiple comparison, ** p < 0.005 (the unedited full‐length immunoblots are presented in the Supplementary Figure ). KCs, keratinocytes; NF‐ĸB, Nuclear Factor kappa B; NTCI, Nuclear Transport Checkpoint Inhibitor; SRTFs, Stress‐Responsive Transcription Factors; STAT3, Signal Transduction and Activator of Transcription 3; TNF‐α, Tumor Necrosis Factor alpha.

Article Snippet: Human Primary Epidermal KCs (ATCC; PCS‐200‐011) used in this study were purchased from American‐Type Culture Collection.

Techniques: Translocation Assay, Incubation, Western Blot, Imaging, Comparison, Transduction

Journal: Skin Health and Disease

Article Title: Anti‐inflammatory control of human skin keratinocytes by targeting nuclear transport checkpoint

doi: 10.1002/ski2.356

Figure Lengend Snippet: NTCI treatment inhibits expression of TSLP and other proinflammatory genes encoding proteins that mediate skin inflammation in cultured human KCs. (a) NTCI suppresses genes encoding the inflammatory mediators in cultured human primary KCs stimulated with TNF‐α/IL‐17A. The gene expression was determined using real‐time quantitative reverse transcription PCR (qRT PCR). The relative expression levels were established with Livak’s methods (2 −∆∆Ct ) with αTubulin gene as the reference and a Mock Control group as the calibrator. The data is presented as a mean + S.E.M. ( n = 4). The statistical significance of the difference between the transcript levels in the cells Stimulated untreated and cells Stimulated treated with the NTCI, cSN50.1 peptide was determined using an ordinary one‐way ANOVA with an uncorrected Fisher’s LSD test for a multiple comparison, ** p < 0.005, *** p < 0.0005. (b) NTCI suppresses production of cognate proteins, TSLP, GM‐CSF (CSF2) and IL‐8 (CXCL8) in cultured human primary KCs stimulated with TNF‐α/IL‐17A. Supernatant samples were collected from the medium of cultured KCs at the indicated timepoints after TNF‐α/IL‐17A stimulation. The proteins concentration was determined in the culture medium using ELISA (TSLP) and CBA (GM‐CSF and IL‐8) assays. The data is presented as mean ± S.E.M. ( n = 4). The statistical significance of the difference between the cytokine/chemokine levels in the media from stimulated cells and stimulated cells treated with the NTCI, cSN50.1 peptide, was determined by a repeated measure two‐way ANOVA using an uncorrected Fisher’s LSD test for multiple comparison, *** p < 0.0005, **** p < 0.0001. CSF2, Granulocyte‐Macrophage Colony‐Stimulating Factor; CXCL8, chemokine IL‐8; KCs, keratinocytes; NTCI, Nuclear Transport Checkpoint Inhibitor; TNF‐α, Tumor Necrosis Factor alpha; TSLP, Thymic Stromal Lymphopoietin.

Article Snippet: Human Primary Epidermal KCs (ATCC; PCS‐200‐011) used in this study were purchased from American‐Type Culture Collection.

Techniques: Expressing, Cell Culture, Gene Expression, Reverse Transcription, Quantitative RT-PCR, Control, Comparison, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Skin Health and Disease

Article Title: Anti‐inflammatory control of human skin keratinocytes by targeting nuclear transport checkpoint

doi: 10.1002/ski2.356

Figure Lengend Snippet: NTCI treatment suppresses time‐dependent production of cytokines and chemokines in cultured human KCs stimulated with PMA. The human primary KCs derived from adult skin were pretreated with NTCI (cSN50.1 peptide) 30 min before stimulation with PMA. Supernatant samples were collected from the medium of cultured KCs at the indicated timepoints after PMA challenge. The proteins concentration in the culture medium were determined using ELISA (TSLP) and CBA (TNF‐α IL‐6, GM‐CSF, and IL‐8) assays. The data is presented as mean ± S.E.M. ( n = 4). The statistical significance of the difference between the cytokine/chemokine levels in the media from stimulated cells and stimulated cells treated with the NTCI, cSN50.1 peptide, was determined by a repeated measure two‐way ANOVA using an uncorrected Fisher’s LSD test for multiple comparison, **** p < 0.0001. CBA, cytometric bead array; ELISA, enzyme‐linked immunosorbent assay; KCs, keratinocytes; NTCI, Nuclear Transport Checkpoint Inhibitor; PMA, Phorbol 12‐Myristate 13‐Acetate; TNF‐α, Tumor Necrosis Factor alpha; TSLP, Thymic Stromal Lymphopoietin.

Article Snippet: Human Primary Epidermal KCs (ATCC; PCS‐200‐011) used in this study were purchased from American‐Type Culture Collection.

Techniques: Cell Culture, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Skin Health and Disease

Article Title: Anti‐inflammatory control of human skin keratinocytes by targeting nuclear transport checkpoint

doi: 10.1002/ski2.356

Figure Lengend Snippet: The mechanism of the NTCI cytoprotective action in inflamed human primary KCs. A mix of cytokines TNF‐α and IL‐17A or skin irritant, PMA, activate the proinflammatory signalling pathways. These pathways are transduced by two SRTFs, NF‐κB RelA and pSTAT3, known to activate proinflammatory and proapoptotic genes. The nuclear translocation of NF‐κB RelA and pSTAT3 is mediated by a nuclear import adaptor complex, Imp α5/Imp β1. The NTCI (cell‐penetrating cSN50.1 peptide) controls this checkpoint. Consequently, selective signalling to the nucleus by SRTFs is stopped and the activation of genes encoding the mediators of inflammation and apoptosis is suppressed [see Suppl. Figure in reference ⁹ ] for more details of this and other signalling pathways as well as the points of action of other anti‐inflammatory therapeutics. KCs, keratinocytes; NF‐ĸB, Nuclear Factor kappa B; NPC, Nuclear Pore Complex; NTCI, Nuclear Transport Checkpoint Inhibitor; PMA, Phorbol 12‐Myristate 13‐Acetate; pSTAT3, phosphorylated STAT3; SRTFs, Stress‐Responsive Transcription Factors; SRTFs, Stress‐Responsive Transcription Factors; TNF‐α, Tumor Necrosis Factor alpha.

Article Snippet: Human Primary Epidermal KCs (ATCC; PCS‐200‐011) used in this study were purchased from American‐Type Culture Collection.

Techniques: Translocation Assay, Activation Assay