Journal: Cell Death & Disease

Article Title: Transglutaminase 2 mediates UV-induced skin inflammation by enhancing inflammatory cytokine production

doi: 10.1038/cddis.2017.550

Figure Lengend Snippet: TG2 is required for the expression of inflammatory cytokines in UV-irradiated keratinocytes. ( a and b ) Skin tissues were obtained from WT and TG2 −/− littermates after 48 h of UV irradiation (200 mJ/cm 2 ). Levels of mRNA ( a ) and protein ( b ) for mouse IL-6 and TNF- α were determined by QRT-PCR and MAGPIX Multiplexing assay, respectively. ( c ) Primary mouse keratinocytes from WT and TG2 −/− littermates were cultured and exposed to UV irradiation (10 mJ/cm 2 ). After 6 h, mRNA levels of mIL-6 and mTNF- α were determined by QRT-PCR ( n =3). ( d and e ) TG2-deficient HaCaT cells were generated by using the CRISPR–Cas9 system (Cas9-TG2) and exposed to UV irradiation (10 mJ/cm 2 ). After 6 h, levels of mRNA ( d , n =3) and secreted protein ( e , n =3) for TNF- α , IL-6, and IL-8 were measured by QRT-PCR and by the CBA method, respectively. n.d., not determined. All data are represented as mean±SEM. *P <0.05; **P <0.01; ***P <0.001

Article Snippet: The cell pellet was resuspended in keratinocyte proliferation media (1 : 1 mixture of KGM-Gold and calcium free KGM-Gold kit media, Lonza, Lyon, France).

Techniques: Expressing, Irradiation, Quantitative RT-PCR, Multiplexing, Cell Culture, Generated, CRISPR

Journal: Cell Death & Disease

Article Title: Transglutaminase 2 mediates UV-induced skin inflammation by enhancing inflammatory cytokine production

doi: 10.1038/cddis.2017.550

Figure Lengend Snippet: UV irradiation activates keratinocyte TG2. ( a and b ) Levels of TG2 protein and in situ TG activity in HaCaT cells were determined by western blot analysis and biotinylated pentylamine (BP) incorporation assay, respectively. HaCaT cells were exposed to UV irradiation ( a , 10 mJ/cm 2 , n =4; b , doses indicated, n =4) and collected at the time indicated ( a ) or after 6 h of UV irradiation ( b ). ( c and d ) mRNA levels of TNF- α , IL-6, and IL-8 were determined by QRT-PCR in HaCaT cells exposed to UV irradiation as ( a ) and ( b ) ( n =3). ( e ) In situ TG activity in the skin from UV-irradiated WT and TG2 −/− littermates ( n =3). ( f ) In situ TG activity in TG2-deficient HaCaT cells (Cas9–TG2) after 6 h of UV irradiation (10 mJ/cm 2 ). All data are represented as mean±SEM. *P <0.05; **P <0.01; ***P <0.001

Article Snippet: The cell pellet was resuspended in keratinocyte proliferation media (1 : 1 mixture of KGM-Gold and calcium free KGM-Gold kit media, Lonza, Lyon, France).

Techniques: Irradiation, In Situ, Activity Assay, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Transglutaminase 2 mediates UV-induced skin inflammation by enhancing inflammatory cytokine production

doi: 10.1038/cddis.2017.550

Figure Lengend Snippet: TG inhibition suppresses the production of cytokines in UV-irradiated keratinocytes. ( a and b ) HaCaT cells were pretreated with KCC009 (250 μ M) or vehicle (Vhcl) 1 h before UV irradiation (10 mJ/cm 2 ). The cells were incubated for 6 h after UV irradiation in the same media. In situ TG activity was measured by the BP incorporation assay ( a , n =3) and visualized by confocal microscopy ( b ). Scale bars, 50 μ m. ( c and d ) Levels of mRNA ( c , n =3) and proteins ( d , n =3) for TNF- α , IL-6, and IL-8 were measured in KCC009-pretreated and UV-irradiated HaCaT cells at the times indicated ( c ) or 6 h after UV irradiation ( d ). ( e ) Levels of TG2 protein and in situ TG activity were measured in HaCaT cell lines expressing pcDNA3, WT (TG2 WT ), or active-site mutant TG2 (TG2 C277S ) established by transfection and selection ( n =4). ( f and g ) Levels of mRNA ( f , n =4) and proteins ( g , n =4) for TNF- α , IL-6, and IL-8 were measured in HaCaT cell lines expressing pcDNA3, WT (TG2 WT ) or active-site mutant TG2 (TG2 C277S ) at 6 h after UV irradiation. n.d., not determined. All data are represented as mean±SEM. *P <0.05; **P <0.01; ***P <0.001

Article Snippet: The cell pellet was resuspended in keratinocyte proliferation media (1 : 1 mixture of KGM-Gold and calcium free KGM-Gold kit media, Lonza, Lyon, France).

Techniques: Inhibition, Irradiation, Incubation, In Situ, Activity Assay, Confocal Microscopy, Expressing, Mutagenesis, Transfection, Selection

Journal: Cell Death & Disease

Article Title: Transglutaminase 2 mediates UV-induced skin inflammation by enhancing inflammatory cytokine production

doi: 10.1038/cddis.2017.550

Figure Lengend Snippet: UV-induced release of ER calcium is responsible for TG2 activation. ( a and b ) HaCaT cells were pretreated with 20 μ M U73122 or U73343 ( a , n =3) and with 100 μ M 2-aminoethyl diphenylborinate (2-APB) ( b , n =3), and then exposed to UV irradiation (10 mJ/cm 2 ). After 6 h, levels of in situ TG activity and mRNA for TNF- α , IL-6, and IL-8 were measured by the BP incorporation assay and QRT-PCR, respectively. ( c and d ) Effect of thapsigargin (500 nM) on in situ TG activity in HaCaT cells ( c , n =3) and the expression of cytokines ( d , n =3) or in primary epidermal keratinocytes prepared from WT and TG2 −/− littermates. All data are represented as mean±SEM. *P <0.05; **P <0.01; ***P <0.001

Article Snippet: The cell pellet was resuspended in keratinocyte proliferation media (1 : 1 mixture of KGM-Gold and calcium free KGM-Gold kit media, Lonza, Lyon, France).

Techniques: Activation Assay, Irradiation, In Situ, Activity Assay, Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: Transglutaminase 2 mediates UV-induced skin inflammation by enhancing inflammatory cytokine production

doi: 10.1038/cddis.2017.550

Figure Lengend Snippet: TG2 enhances the transcriptional activity of NF- κ B. ( a – c ) 3 κ B-luciferase reporter activity was assessed in primary epidermal keratinocytes prepared from WT and TG2 −/− littermates ( a , n =3) or HaCaT cells ( b and c ; n =3) 9 h after UV irradiation (10 mJ/cm 2 ). HaCaT cells were pretreated with 250 μ M KCC009 ( b ), or with 20 μ M U73122 or U73343 ( c ) 1 h before UV irradiation. Luciferase activity was normalized with co-transfected Renilla activity. Data are represented as mean±SEM. *P <0.05; **P <0.01; ***P <0.001. ( d and e ) Lysates prepared from TG2 knockdown HaCaT cells ( d ) or HaCaT cells treated with 250 μ M KCC009 ( e ) were immunoblotted with antibodies specific for I κ B α , phosphorylated p65 at Ser536, p65, and TG2. Actin was used as a loading control. An arrowhead in the blot indicates a nonspecific band. ( f ) Schematic representation of TG2-dependent UV-induced inflammation

Article Snippet: The cell pellet was resuspended in keratinocyte proliferation media (1 : 1 mixture of KGM-Gold and calcium free KGM-Gold kit media, Lonza, Lyon, France).

Techniques: Activity Assay, Luciferase, Irradiation, Transfection, Knockdown, Control