Supplemental data For Allen et. al

Supplemental data for Allen et. al submitted to the Journal of Neurophysiolog

Effect of Variations in the Alkyl Chain Lengths of Self-Assembled Monolayers on the Crystalline-Phase-Mediated Electrical Performance of Organic Field-Effect Transistors

Self-assembled monolayers (SAMs) of organic molecules are frequently employed to improve the electrical performance of organic field-effect transistors (OFETs). However, the relationship between SAM properties and OFET performance has not been fully explored, leading to an incomplete understanding of the system. This study investigates the effect of the SAM alkyl chain length on the crystalline phase of pentacene films and OFET performance. Two types of SAMswith alkyl chain lengths of 10 (decyltrichlorosilane, DTS) and 22 (docosyltrichlorosilane, DCTS)were examined, and variations in the performance of pentacene-based OFETs with the nature of the SAM treatment were observed. Despite the similar surface morphologies of the pentacene films, field-effect mobility in the DCTS-treated OFET was twice that in the DTS-treated OFET. To find the reason underlying the dependence of the OFET’s electrical performance on the SAM alkyl chain length, X-ray diffraction measurements were conducted, followed by a phase analysis of the pentacene films. Bulk and thin-film phases were observed to coexist in the pentacene film grown on DTS, indicating several structural defects in the film; this can help explain the dependence of the OFET electrical performance on the SAM alkyl chain length, mediated by the different crystalline phases of pentacene

The NBD of NOD2 interacts with both TRAF6 and UBA domains of p62.

<p><b>A</b>. NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. <b>B</b>. HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). <b>C</b>. Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.</p

p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

<div><p>NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-κB activation and p38 MAPK, and subsequent production of cytokines IL-1β and TNF-α. p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.</p> </div

Synthesis of Cs₃MnBr₅ Green Phosphors Using an Eco-Friendly Evaporative Crystallization Process

Green (G) and red (R) light-emitting materials, such as quantum dots, perovskite nanocrystals, and inorganic phosphor powders, owing to their excellent optical characteristics, have attracted researchers’ attention as color-conversion materials for lighting and display applications. However, these materials contain environmentally harmful elements, such as Pb or Cd, and/or they are synthesized using environmentally harmful synthetic approaches and conditions, involving the use of organic solvents, high pressure, high temperature, harsh atmosphere, and long reaction time. In this study, as an eco-friendly synthetic approach to synthesize lead-free Cs3MnBr5 G powder phosphor, we suggest an evaporative crystallization process of aqueous reactant solution. This synthetic process does not use toxic elements or solvents and the crystallization process utilizes only low reaction temperature and short reaction time under air atmosphere conditions. We successfully synthesized Cs3MnBr5 green powder phosphor, with excellent optical properties, by evaporative heating of a 200 nm syringe-filtered solution at 150 °C for 2 h. The synthesized Cs3MnBr5 phosphors have a photoluminescence quantum yield of 66.3%, a peak wavelength of 520 nm, a narrow bandwidth of 38 nm, and a photoluminescence decay time of 0.34 ms under blue excitation. This phosphor is expected to be a useful alternative G-emitting material that can compete with commercial green quantum dots, perovskite nanocrystals, or inorganic phosphors

p62 co-localizes with NOD2 through the NBD domain of NOD2.

<p><b>A</b>. HEK293T cells were transfected for 24 h with scramble siRNA (left top panel), si-p62 (right top panel), and GFP-NOD2. GFP-NOD2 was visualized using confocal microscopy as described in “Methods”. <b>B</b>. Similarly, DsRed-NBD domain, LRR region or full-length NOD2 and GFP-p62 expression vectors were transfected in HEK293T cells and co-localization of these proteins was examined using confocal microscopy. <b>C</b>. Immunogold staining of co-localized pCMV-HA-p62 (18 nm colloidal gold) and GFP-NOD2 (10 nm colloidal gold) in HEK293T cells. Cells on grids were viewed using a transmission electron microscope. Scale bars: 500 nm (left bottom), 100 nm (middle, right bottom). <b>D</b>. HEK293T cells were transfected with DsRed-NOD2 and GFP-LC3 plasmids. Cells were observed by confocal microscopy and images were acquired using ZEN software.</p

p62 stabilizes gMDP-induced NOD2 oligomers.

<p><b>A</b>. HEK293T cells were stably transfected with pLNCX-NOD2 as described in “Methods”. These cells were treated with scramble (si-Scramble) or p62 targeting (si-p62) small interference RNAs for 24 h. Cells were then treated with the translation inhibitor cyclohexamide (CHX, 100 µg/ml) and gMDP (5 µg/ml) for the time indicated, and immunoblots against NOD2 were performed. Intensities of NOD2 bands in comparison with p38 bands (loading control) were expressed as 100% for control samples (right panel). The ImageJ (NIH) program was used for densitometry analysis and data were expressed as mean ± S.D. (n≥4). *p<0.05 (Student t-test). <b>B</b>. HEK293T cells were transiently transfected with Myc-NOD2, HA-NOD2, and scramble (si-Scramble) or p62-targeting (si-Scramble) small interference RNAs. Myc-NOD2 was immunoprecipitated with anti-Myc antibodies and immunoblots were performed against HA or Myc. Immunoblots for total lysates against HA and p62 were performed for HA-NOD2 and endogenous p62 inputs (3^rd and 4^th lanes, respectively). <b>C</b>. HEK293T cells were transfected with Myc-NOD2 at 16 h post-transfection with scrambled siRNA or p62-siRNA. After 24 h, cells were further cultured without or with gMDP (5 µg/ml) for 4 h and cell extracts were loaded onto the Superdex™ 200 column. Fractions were analyzed by immunoblot using Myc antibody for estimation of Myc-NOD2 oligomerization (upper panel). Immunoblot for p38 was used as a control. Myc-NOD2 and p38 immunoreactivities were analyzed using NIH ImageJ program (bottom panel).</p

NOD2 physically interacts with p62.

<p><b>A–C</b>. HEK293T cells were transiently transfected with expression vectors encoding GFP-tagged p62 (GFP-p62) and/or Myc-tagged NOD2 (Myc-NOD2). After 24 h, total cell lysates were subjected to immunoprecipitation using anti-GFP (A) or anti-Myc (B) antibodies and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against GFP and HA. <b>C</b>. Similar experiments as A-B were performed but with or without N-glycorylated muramyldipeptide (gMDP: 5 µg/mL) treatments for 4 h. Data shown are representative images of 3 independent experiments.</p

p62 enhances pro-IL-1β expression and TNF-α production in macrophages.

<p><b>A</b>. RAW 264.7 cells were transfected with scrambled (si-Scramble) or p62 targeting (si-p62) small interference RNAs using Lipofectamine™ 2000. Twenty four hours post-transfection, cells were treated with a low dose of LPS (50 ng/mL) for 4 h, rinsed with complete media twice, and then incubated with gMDP (5 µg/mL) for another 4 h. Expression of pro-IL-1β was detected using an antibody against IL-1β and p38 was used as a loading control. Densitometric analysis of blots was done using ImageJ (NIH). Data are expressed as mean ± S.D. (n = 3). N.S., not significant; * p<0.05 (Student t-test). <b>B</b>. RAW 264.7 cells were transfected with scramble siRNA or p62-siRNA as above A, and cells were treated with LPS (20 ng/mL) for 4 h. After two washes with complete media, cells were further incubated with gMDP (5 µg/mL) for another 4 h and TNF-α concentrations in cell culture media were measured by ELISA according to the manufacturer's instructions (eBioscience). Data are expressed as mean ± S.D. (n = 4); * p<0.05 (Tukey's Multiple Comparison Test). <b>C</b>. THP-1 cells were stably transfected with sh-scramble (sh-Scramble) or p62 targeting (sh-p62) small-hairpin RNA producing constructs as described in “Methods”. Three cell clones stably knocked down in p62, pooled sh-Scramble control clones and non-treated wild-type cells were treated with gMDP (5 µg/mL) for 4 h and TNF-α production in the cell culture media was measured using TNF-α bioassay as described in “Methods”.</p

p62 is required for the activation of NF-κB and p38 MAPK, and ubiquitination of RIP2 and TRAF6.

<p><b>A</b>. HEK293T cells were transfected with pCMV-Myc-NOD2 and NF-κB luciferase reporter constructs in the presence of scrambled or p62-targeting small interference RNAs (si-p62). Cells were then treated with gMDP (5 µg/mL) for 4 h and NF-κB activity was measured. Data are expressed as the fold of luciferase activity ± SD (n = 3). <b>B-C</b>. HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then transfected with expression vectors for HA-ubiquitin (HA-Ub) and pcDNA3-Myc-RIP2 or pCMV-Myc-TRAF6 for another 24 h. After treating the cells with gMDP (5 µg/mL) for 4 h, RIP2 (B) or TRAF6 (C) was immunoprecipitated with Myc antibodies from total cell lysates and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against HA. Myc-RIP2 or Myc-TRAF6, NOD2 and HA-ubiquitin were analyzed by immunoblot as the inputs (bottom panels). p62 protein levels were also measured by immunoblot. Data shown are representative images of 3 independent experiments. <b>D</b>, HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then treated with gMDP (5 µg/ml) for the times indicated. Activation of p38 was analysed through immunoblotting against tyrosine phosphoryled p38. The ImageJ (NIH) program was used for densitometry analysis of phosphor-p83 bands and data were expressed as mean ± S.D. (n = 3).</p