1. MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation 
Anja Schmitt, Paula Grondona, Tabea Maier, Marc Brändle, Caroline Schönfeld, Günter Jäger, Corinna Kosnopfel, Franziska C. Eberle, Birgit Schittek, Klaus Schulze-Osthoff, Amir S. Yazdi, Stephan Hailfinger 
Journal of Investigative Dermatology 
Volume 136, Issue 4, Pages (April 2016)
DOI: /j.jid
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2. Figure 1
Activation of PKC leads to MALT1 protease activity in KCs. (a) In vitro LVSR-amc cleavage activity of human primary KCs stimulated for 30 minutes with PMA or PMA/I (P/I). (b) Pretreatment with the MALT1 inhibitor LVSR-fmk for 30 minutes. (c) LVSR-amc cleavage activity was normalized to the protein concentration of the lysate. (d) Immunoblot analysis of primary KCs that have been pretreated with the proteasome inhibitor MG-132 for 15 minutes and subsequently stimulated with P/I for 1 hour as indicated. Immunoblot analysis of primary KCs that have been pretreated with the MALT1 inhibitor LVSR-fmk for 30 minutes and subsequently stimulated with P/I for (e) 1 hour or (f) 12 hours as indicated. Cleavage products are indicated by arrowheads. Error bars correspond to mean ± SD. Statistical significance was calculated using a paired t-test (***P < 0.01). Data are representative of three (a–e) or two (f) independent experiments. KCs, keratinocytes; MALT1, mucosa-associated lymphoid tissue lymphoma translocation gene 1; P/I, PMA/ionomycin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SD, standard deviation.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions

3. Figure 2
MALT1 protease activity depends on a novel PKC isoform, CARMA2 and BCL10. (a) Immunoblot analysis of human primary KCs that have been stimulated with PMA for 1 hour as indicated. (b) KCs have been pretreated with STN or Gö6976 for 30 minutes and stimulated with P/I for 1 hour as indicated. (c, d) Human primary KCs were electroporated with scrambled (scr), CARMA2- (siCARMA2), or BCL10- (siBCL10) targeting siRNA and stimulated after 4 days with P/I for 1 hour. Cleavage of RelB was analyzed by western blot. (a–d) KCs were treated with MG-132 for 15 minutes before stimulation. Data are representative of three independent experiments. KCs, keratinocytes; P/I, PMA/ionomycin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; STN, sotrastaurin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions

4. Figure 3
Zymosan and S. aureus activate MALT1 protease activity. (a) In vitro LVSR-amc cleavage activity of KCs that were stimulated for 30 minutes as indicated. (b) LVSR-amc cleavage activity was normalized to the protein concentration of the lysate. (c) KCs were lentivirally transduced with shRNA targeting MALT1 and stimulated as indicated. (d) Immunoblot analysis of primary KCs that have been pretreated with either LVSR-fmk or Src kinase inhibitor PP2 for 30 minutes and subsequently stimulated with zymosan for 1 hour as indicated. (e) KCs were electroporated with scrambled (scr) or Dectin-1 (siDectin-1) targeting siRNA and stimulated after 3 days with P/I for 1 hour. (f) KCs were pretreated with STN or Gö6976 for 30 minutes and stimulated with zymosan for 1 hour as indicated. (g) KCs were pretreated with either LVSR-fmk or Src kinase inhibitor PP2 for 30 minutes and subsequently stimulated with live S. aureus for 1 hour as indicated. (c–g) KCs were treated with MG-132 for 15 minutes before stimulation. Cleavage products are indicated by arrowheads. Error bars correspond to mean ± SD. Data are representative of three independent experiments. Statistical significance was calculated using a paired t-test (***P < 0.01; **P < 0.05). KCs, keratinocytes; shRNA, small hairpin RNA; siRNA, small interfering RNA; SD, standard deviation; STN, sotrastaurin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Identification of target genes regulated by MALT1 protease activity in human primary KCs. Global gene expression analysis of primary human KCs treated with either solvent control, P/I, or P/I in combination with MALT1 inhibitor for 8 hours. (a) Identification of a group of genes that were upregulated by P/I stimulation, but were unaffected by the MALT1 inhibitor or (b) were reduced in expression upon MALT1 inhibition. (c) Heat map of MALT1 inhibitor-sensitive genes. (a–c) Sample numbers correspond to biological triplicates. KCs, keratinocytes; P/I, PMA/ionomycin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
MALT1 protease activity controls the expression of inflammatory genes. (a) Gene expression profiling of MALT1-sensitive target genes by qPCR of human primary KCs treated with P/I alone or P/I in combination with MALT1 inhibitor for 8 hours. Expression was normalized to GAPDH levels. (b, c) KCs were pretreated with LVSR-fmk for 30 minutes and stimulated either with PMA, P/I, or zymosan for 16 hours as indicated. Supernatants were analyzed for secreted (b) TNFα or (c) CXCL8 by ELISA. (d, e) MALT1 silenced KCs were stimulated and measured by ELISA as in (b, c). Error bars correspond to mean ± SD. Data are representative of three independent experiments. Statistical significance was calculated using a paired t-test (***P < 0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KCs, keratinocytes; P/I, PMA/ionomycin; PMA, phorbol 12-myristate 13-acetate; TNFα, tumor necrosis factor-α; qPCR, quantitative PCR; SD, standard deviation.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
MALT1 protease activity is essential for bacteria killing upon P/I or zymosan stimulation. (a) Conditioned media were generated by pretreatment with MALT1 inhibitor for 30 minutes and subsequent stimulation of human primary KCs with PMA for 18 hours. Nonstimulated human primary KCs were incubated with the conditioned supernatants for 24 hours, and expression of the indicated genes was analyzed by qPCR. (b) KCs were pretreated with LVSR-fmk for 30 minutes and stimulated with either P/I or zymosan for 24 hours as indicated. S. aureus was incubated with lysates of the stimulated KCs for 2 hours at 37°C, plated overnight on LB agar and colony numbers were quantified. Error bars correspond to mean ± SD. Statistical significance was calculated using a paired t-test (***P < 0.01; **P < 0.05). Data are representative of (a) two or (b) three independent experiments. KCs, keratinocytes; P/I, PMA/ionomycin; PMA, phorbol 12-myristate 13-acetate; qPCR, quantitative PCR; SD, standard deviation.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions