Journal: Aging (Albany NY)

Article Title: Ambient particulate matter attenuates Sirtuin1 and augments SREBP1-PIR axis to induce human pulmonary fibroblast inflammation: molecular mechanism of microenvironment associated with COPD

doi: 10.18632/aging.102077

Figure Lengend Snippet: Signatures regulated by PM in HPF models. ( A ) The mRNA level of SREBP1 after PM treatment. ( B ) The prediction of SREBP1 -network derived from the common signature, by comparison of IPA database with microarray data from HPF cells with a 1.5-fold-change cut-off. The intensity of the node color indicates the degree of activating (orange) or inhibiting (blue) regulation following PM interaction. ( C ) Several mRNA levels of SREBP1 downstream factors, including PIR, LIF, IL-6, IL-24, and CXCL8, after PM treatment in HPF.

Article Snippet: Western blot analysis was performed with primary anti-PIR antibody (GeneTex, Irvine CA, USA), anti-NLRP3 antibody (GeneTex, Irvine, CA, USA), anti-SREBP1 antibody (GeneTex, Irvine CA, USA), anti-IL-6 antibody (Abcam, Cambridge MA, USA), anti-IL-1β antibody (GeneTex, Irvine, CA, USA), anti-SIRT1 antibody (Proteintech®, Rosemont, USA), and anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Derivative Assay, Comparison, Microarray

Journal: Aging (Albany NY)

Article Title: Ambient particulate matter attenuates Sirtuin1 and augments SREBP1-PIR axis to induce human pulmonary fibroblast inflammation: molecular mechanism of microenvironment associated with COPD

doi: 10.18632/aging.102077

Figure Lengend Snippet: PM exposure is positively correlated with activation of the SREBP1-PIR signaling pathway through SIRT1 downregulation. ( A ) Western blot analysis of PIR after PM treatment in HPF. ( B ) PIR activity from cell culture supernatant in PM-induced models. ( C ) Levels of SREBP1, SIRT1, and PIR were measured from cell culture supernatant of PM-induced models. ( D ) mRNA levels of SREBP1 in PF429242-PM co-treated HPF models. ( E ) mRNA levels of PIR in PF429242-PM co-treated HPF models. ( F ) PIR activity from cell culture supernatant in PF429242-PM co-treated HPF models. ( G ) Western blot analysis of PIR and SIRT1 in TPh A-PM co-treated HPF models. ( H ) PIR activity from cell culture supernatant in TPh A-PM co-treated HPF models. ( I ) mRNA level of PIR in Ex527-PM co-treated HPF models. ( J ) Western blot analysis of SIRT1 and PIR in Ex527-PM co-treated HPF models. ( K ) Level of PIR was measured from cell culture supernatant in Ex527-PM co-treated HPF models.

Article Snippet: Western blot analysis was performed with primary anti-PIR antibody (GeneTex, Irvine CA, USA), anti-NLRP3 antibody (GeneTex, Irvine, CA, USA), anti-SREBP1 antibody (GeneTex, Irvine CA, USA), anti-IL-6 antibody (Abcam, Cambridge MA, USA), anti-IL-1β antibody (GeneTex, Irvine, CA, USA), anti-SIRT1 antibody (Proteintech®, Rosemont, USA), and anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Activation Assay, Western Blot, Activity Assay, Cell Culture

Journal: Aging (Albany NY)

Article Title: Ambient particulate matter attenuates Sirtuin1 and augments SREBP1-PIR axis to induce human pulmonary fibroblast inflammation: molecular mechanism of microenvironment associated with COPD

doi: 10.18632/aging.102077

Figure Lengend Snippet: NLRP3 inflammasome was upregulated by PM exposure. ( A ) mRNA level of NLRP3 after PM in HPF. ( B ) Western blot analysis of NLRP3 and IL-1β after PM in HPF. ( C ) mRNA level of NLRP3 in Ex527-PM co-treated HPF models. ( D ) mRNA levels of NLRP3 in PF429242-PM co-treated HPF models. ( E ) Western blot analysis of PIR, SREBP1, and NLRP3 in TPh A-PM co-treated HPF models.

Article Snippet: Western blot analysis was performed with primary anti-PIR antibody (GeneTex, Irvine CA, USA), anti-NLRP3 antibody (GeneTex, Irvine, CA, USA), anti-SREBP1 antibody (GeneTex, Irvine CA, USA), anti-IL-6 antibody (Abcam, Cambridge MA, USA), anti-IL-1β antibody (GeneTex, Irvine, CA, USA), anti-SIRT1 antibody (Proteintech®, Rosemont, USA), and anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot

Journal: Aging (Albany NY)

Article Title: Ambient particulate matter attenuates Sirtuin1 and augments SREBP1-PIR axis to induce human pulmonary fibroblast inflammation: molecular mechanism of microenvironment associated with COPD

doi: 10.18632/aging.102077

Figure Lengend Snippet: A model illustrates that PM activates the SREBP1-PIR/inflammasomes signaling axis through SIRT1 -mediated modulation. ( A ) A schematic model to describe the positive feedback loop and detailed mechanisms of the SIRT1-SREBP1-PIR axis and crosstalk with NF-κB signaling. ( B ) SIRT1 modulates the SREBP1-PIR/NLRP3 inflammasome axis in response to PM. SIRT1 functions as a protector from PM exposure. The SIRT1-SREBP1-PIR/NLRP3 inflammasome axis may present an attractive therapeutic target for PM-related adverse health events.

Article Snippet: Western blot analysis was performed with primary anti-PIR antibody (GeneTex, Irvine CA, USA), anti-NLRP3 antibody (GeneTex, Irvine, CA, USA), anti-SREBP1 antibody (GeneTex, Irvine CA, USA), anti-IL-6 antibody (Abcam, Cambridge MA, USA), anti-IL-1β antibody (GeneTex, Irvine, CA, USA), anti-SIRT1 antibody (Proteintech®, Rosemont, USA), and anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA).

Techniques:

Journal: bioRxiv

Article Title: PacC-dependent adaptation and modulation of host cellular pH controls hemibiotrophic invasive growth and disease development by the rice blast fungus

doi: 10.1101/2020.06.22.164590

Figure Lengend Snippet: (A) Expression of PAG1 in mycelium of WT P131, Δ pacC , NGP239, and NGP559. For the Q-PCR, the expression level of P131 cultured at pH 5.5 was arbitrarily set to 1. (B) PacC binds to GCCAAG motifs in the PAG1 promoter. Purified GST-PacC 222 protein was used to detect binding of putative PacC binding motifs. Probes 1 and 2 contain predicted PacC-binding motifs and were prepared by labelling with 32 P-dCTP and incubated with GST-PacC 222 for 30 min, before loading a native-PAGE gel. For competition experiments, 100x or 10x concentrations of un-labelled Probe 1 were mixed with the GST-PacC 222 protein for 30 min before incubation with 32 P-dCTP-labelled probes. (C) WT and Δ pag1 are same in colony growth on OTA plates. (D) WT and Δ pag1 are equivalent in conidiation. (E) Reduced virulence of Δ pag1 mutant compared to WT. (F) Infection assays of WT and Δ pag1 on barley epidermis. (G) Arrested biotrophic growth of Δ pag1 compared to WT. Bar = 25 μm.

Article Snippet: GST-fused PacC 559 , PacC 222 and PacC 80 proteins were individually expressed in the pGEX-4T-3 vector (Promega, USA) in E. coli BL21DE3 and purified, as described in supplementary data.

Techniques: Expressing, Cell Culture, Purification, Binding Assay, Incubation, Clear Native PAGE, Mutagenesis, Infection

Journal: bioRxiv

Article Title: PacC-dependent adaptation and modulation of host cellular pH controls hemibiotrophic invasive growth and disease development by the rice blast fungus

doi: 10.1101/2020.06.22.164590

Figure Lengend Snippet: (A) Infected plant cells are alkalinized during the early biotrophic growth of M. oryzae and then become acidified during the later necrotrophic growth. (B) During biotrophic growth, the PacC 559 and PacC 222 transcription factor isoforms localize to the nucleus, where PacC 559 acts as a transcriptional repressor to repress expression of genes associated with conidiation and necrotrophic growth, including PRG1 , HTF1 , and PIG1 while PacC 222 acts as a transcriptional activator to activate genes associated with biotrophic growth. (C) As host cells become acidified and lose viability, the PacC functional isoforms exit from the nucleus thereby de-repressing expression of genes related to necrotrophic growth and conidiation.

Article Snippet: GST-fused PacC 559 , PacC 222 and PacC 80 proteins were individually expressed in the pGEX-4T-3 vector (Promega, USA) in E. coli BL21DE3 and purified, as described in supplementary data.

Techniques: Infection, Expressing, Functional Assay

Journal:

Article Title: YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

doi: 10.1128/MCB.23.5.1647-1655.2003

Figure Lengend Snippet: Previously undescribed mutant pacC alleles characterized in this work

Article Snippet: N-terminally His-tagged PacC proteins (wild-type, double-mutant Y455D-Y662N, and mutant L340S) were synthesized in vitro and labeled with [ 35 S]methionine (1,000 Ci/mmol) by using the Promega TNT coupled transcription-translation system and appropriate templates (Table ).

Techniques: Mutagenesis, Sequencing

Journal:

Article Title: YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

doi: 10.1128/MCB.23.5.1647-1655.2003

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: N-terminally His-tagged PacC proteins (wild-type, double-mutant Y455D-Y662N, and mutant L340S) were synthesized in vitro and labeled with [ 35 S]methionine (1,000 Ci/mmol) by using the Promega TNT coupled transcription-translation system and appropriate templates (Table ).

Techniques: Plasmid Preparation

Journal:

Article Title: YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

doi: 10.1128/MCB.23.5.1647-1655.2003

Figure Lengend Snippet: Two-hybrid interaction of PalA with PacC. Yeast strain CTY10-5d was used, and proteins were expressed from plasmids listed in Table ​Table2.2. (A) GAD-PacC fusions contain the indicated PacC residues. The shaded bar indicates the DNA binding domain (DBD). Arrows mark the approximate position of the signaling-protease (∼493 to 500) (8) and processing-protease (∼252 to 254) (25) cleavage sites. Values are the average β-galactosidase activity of four transformants. Standard errors were <14%. In control experiments, GAD protein fusions did not interact with LexA (<0.4 U). ND, not determined. (B) Western analysis of protein extracts from transformants expressing LexA-PalA and the indicated GAD-PacC protein fusions which were detected with anti-HA antibodies. WT, wild type.

Article Snippet: N-terminally His-tagged PacC proteins (wild-type, double-mutant Y455D-Y662N, and mutant L340S) were synthesized in vitro and labeled with [ 35 S]methionine (1,000 Ci/mmol) by using the Promega TNT coupled transcription-translation system and appropriate templates (Table ).

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing

Journal:

Article Title: YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

doi: 10.1128/MCB.23.5.1647-1655.2003

Figure Lengend Snippet: In vitro binding of PalA to PacC by using pull-down assays. (A) GST fusion proteins bound to glutathione-Sepharose 4B beads (Pharmacia) used in the binding assays (10% of the total) were separated by SDS-PAGE (10% polyacrylamide) and stained with Coomassie blue. (B) Beads loaded with GST alone (lanes 4 to 6) or with GST fusions to PalA (lanes 7 to 9), PacC(529-678) (lanes 10 to 12), or PacC (169-410) (lanes 13 and 14) were incubated with in vitro-synthesized [35S]PacC (wild type, double-mutant Y455D-Y662N, or mutant L340S, as indicated). After being washed, bound proteins were boiled in sample buffer, separated by SDS-PAGE, and analyzed by autoradiography (top) and by Coomassie staining (bottom). Lanes marked Input contain in vitro-synthesized PacC (wild type [lane 1], double-mutant Y455D-Y662N [lane 2] and mutant L340S [lane 3]) used for binding experiments (20% of the total reaction mixture). Protein markers are in kilodaltons.

Article Snippet: N-terminally His-tagged PacC proteins (wild-type, double-mutant Y455D-Y662N, and mutant L340S) were synthesized in vitro and labeled with [ 35 S]methionine (1,000 Ci/mmol) by using the Promega TNT coupled transcription-translation system and appropriate templates (Table ).

Techniques: In Vitro, Binding Assay, SDS Page, Staining, Incubation, Synthesized, Mutagenesis, Autoradiography