Journal: Cell Host & Microbe

Article Title: Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway

doi: 10.1016/j.chom.2023.02.002

Figure Lengend Snippet:

Article Snippet: HCP216549-SG01-3 for generation of p11-KO cell line , GeneCopoiea , Cat# HCP216549-SG01-3.

Techniques: Virus, Recombinant, Cell Culture, Protease Inhibitor, Transfection, SYBR Green Assay, Mass Spectrometry, CyQUANT Assay, LDH Cytotoxicity Assay, cDNA Synthesis, Purification, Gene Expression, Construct, Control, Transformation Assay, Cloning, Plasmid Preparation, Expressing, Software, Imaging, Blocking Assay, Red Blood Cell Lysis, Membrane

Journal: eLife

Article Title: TIGAR deficiency enhances skeletal muscle thermogenesis by increasing neuromuscular junction cholinergic signaling

doi: 10.7554/eLife.73360

Figure Lengend Snippet: ( A ) Representative images of α-bungarotoxin immunofluorescent labeling of nicotinic acetylcholine receptor clusters in the extensor digitorium longus (EDL) muscle from wild type (WT) and whole-body Tigar knockout (TKO) mice. ( B ) Quantification of the number of nicotinic acetylcholine receptor clusters following 15 min exposure at 4°C (WT n = 17, TKO n = 13). These data represent the average of over six mice in each group of mean ± SD (unpaired t -test, two-tailed, **p=0.0035). ( C ) Acetylcholine levels in the gastrocnemius muscle of WT and TKO male (n = 7) mice at room temperature or following 1 hr at 4°C. ( D ) Acetylcholine levels in the gastrocnemius muscle of Chat Cre and chTKO male (n = 6) mice at room temperature or following 1 hr at 4°C. ( E ) Representative immunoblots of TIGAR and actin proteins from two scrambled sgRNA and two Tigar sgRNA knockout Sh-SY5Y cell lines. ( F ) Acetylcholine concentrations in the medium of scrambled and TKO SH-SH5Y neuroblastoma cells. ( G ) m2 acetylcholine enrichments in cells labeled with d -glucose-1,2- 13 C 2. ( H ) m2 acetylcholine enrichments in cells labeled with U- 13 C 6 d -glucose. ( I ) m1 glutamate ( m/z 128, C2-C4 fragment) enrichment in the medium of scrambled and TKO SH-SY5Y human neuroblastoma cells labeled with d -glucose-[1,2]- 13 C2 . Statistical analyses are described in ‘Method details,’ and the data are presented as the mean ± SD. *p<0.05, ***p<0.001, ****p<0.0001. Figure 6—source data 1. The culture SH-SY5Y cells were collected from both scrambled and whole-body Tigar knockout (TKO) cells, and 30 μg of the cell lysates were used for TIGAR (30 kDa) immunoblotting analysis as described in the ‘Immunoblotting’ section. The left four lanes of the raw image (lysate from neuroblastoma cells) were used for to confirm the efficiency of TIGAR protein loss in the SH-SY5Y neuroblastoma TKO cells. The right four lanes of the raw image represent the TIGAR immunoblotting of the cell lysates from 7-day differentiated neuroblastoma cells. A detailed description of the raw images is shown in . Figure 6—source data 2. The culture SH-SY5Y cells were collected from both scrambled and whole-body Tigar knockout (TKO) cells, and 30 μg of the cell lysates were used for actin β (42 kDa) immunoblotting analysis as described in the ‘Immunoblotting’ section. The left four lanes of the raw image (lysate from neuroblastoma cells) were used for to show actin β protein in the SH-SY5Y neuroblastoma cells. The right four lanes of the raw image represent the actin β immunoblotting of the cell lysates from 7-day differentiated neuroblastoma cells. A detailed description of the raw images is shown in .

Article Snippet: The CRISPR 3xsgRNA/Cas9 all-in-one expression clone targeting Tigar (NM_020375.2) (Cat# HCP215394-CG04-3) and scrambled sgRNA control for pCRISPR-CG04 (Cat# CCPCTR01-CG04-B) were purchased from GeneCopoeia (Rockville, MD).

Techniques: Labeling, Knock-Out, Two Tailed Test, Western Blot

Journal: eLife

Article Title: TIGAR deficiency enhances skeletal muscle thermogenesis by increasing neuromuscular junction cholinergic signaling

doi: 10.7554/eLife.73360

Figure Lengend Snippet:

Article Snippet: The CRISPR 3xsgRNA/Cas9 all-in-one expression clone targeting Tigar (NM_020375.2) (Cat# HCP215394-CG04-3) and scrambled sgRNA control for pCRISPR-CG04 (Cat# CCPCTR01-CG04-B) were purchased from GeneCopoeia (Rockville, MD).

Techniques: Knock-Out, Recombinant, Expressing, Control, Sequencing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Lysis, Blocking Assay, Electron Microscopy, Software

Journal: JCI Insight

Article Title: PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis

doi: 10.1172/jci.insight.125326

Figure Lengend Snippet: (A and B) Upregulation of immune checkpoint CD274 and PDCD1LG2 in invasive lung fibroblasts. RNA-seq (n = 9 per group) (A) and qRT-PCR analysis (n = 6 per group) (B) of CD274 and PDCD1LG2 expression in invasive and noninvasive IPF lung fibroblasts. (C) Cell surface expression of CD274 and PDCD1LG2 in invasive and noninvasive IPF lung fibroblasts. (D) Single-cell Western blot analysis of CD274 expression in invasive and noninvasive lung fibroblasts. (E) Cell surface expression of CD274 and PDCD1LG2 in primary IPF fibroblasts and healthy controls by flow cytometry. (F) Flow cytometry analysis of lung single-cell homogenate for CD274 expression in CD31–CD45–EPCAM– cells from IPF (n = 7) or healthy (n = 6) samples. Throughout, data are the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by 1-way ANOVA (A, B, and E) or Student’s t test (F).

Article Snippet: For CD274-KO, we first generated a Cas9-expressing cell line (Invitrogen LentiArray Cas9 Lentivirus, {"type":"entrez-nucleotide","attrs":{"text":"A32069","term_id":"1249524"}} A32069 , Thermo Fisher Scientific), and then sgRNA expression clones targeting CD274 (HCP208443-SG01-3-10, GeneCopoeia) and scrambled sgRNA control plasmid (CCPCTR01-SG01-10, GeneCopoeia) were used to generate CD274-KO and control cells.

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Single Cell Western, Flow Cytometry

Journal: JCI Insight

Article Title: PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis

doi: 10.1172/jci.insight.125326

Figure Lengend Snippet: (A–D) CD274 in control (CTL) and CD274-KO IPF lung fibroblasts was assessed by Western blot analysis along with GAPDH (A), and cell surface expression along with PDCD1LG2 (B), and the fibroblasts evaluated for migration and invasion (C and D). (E–H) CD274 in CTL and CD274-activated IPF lung fibroblasts was assessed by Western blot analysis along with GAPDH (E), cell surface expression along with PDCD1LG2 (F), and the fibroblasts evaluated for migration and invasion (G and H). GAPDH served as loading control. See complete unedited blots in the supplemental material. Representative images of migration and invasion are shown. Scale bars: 1 mm. Data are the mean ± SEM (n = 3 per group). *P < 0.05 by Student’s t test.

Article Snippet: For CD274-KO, we first generated a Cas9-expressing cell line (Invitrogen LentiArray Cas9 Lentivirus, {"type":"entrez-nucleotide","attrs":{"text":"A32069","term_id":"1249524"}} A32069 , Thermo Fisher Scientific), and then sgRNA expression clones targeting CD274 (HCP208443-SG01-3-10, GeneCopoeia) and scrambled sgRNA control plasmid (CCPCTR01-SG01-10, GeneCopoeia) were used to generate CD274-KO and control cells.

Techniques: Control, Western Blot, Expressing, Migration

Journal: JCI Insight

Article Title: PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis

doi: 10.1172/jci.insight.125326

Figure Lengend Snippet: Gene expression (n = 3 per group) (A) and Western blot analysis (B) of CD274, PDCD1LG2, TP53, and GAPDH in IPF lung fibroblasts treated with Si-CTL, Si-CD274, Si-PDCD1LG2, or Si-TP53. See complete unedited blots in the supplemental material. Cell surface expression (C) of CD274 and PDCD1LG2 in IPF lung fibroblasts treated with Si-CTL, Si-CD274, Si-PDCD1LG2, or Si-TP53 after 68 hours. (D) Representative cell growth curve of lung fibroblasts treated with Si-CTL, Si-CD274, or Si-PDCD1LG2. (E) Representative images of lung fibroblasts treated with Si-CTL, Si-CD274, or Si-PDCD1LG2 after 68 hours. Scale bar: 150 μm. (F and G) In vitro migration and invasion assay. Equal numbers of cells were seeded in the upper part of transwells. (F) Representative images of migrated and invasive Si-CTL or Si-TP53 lung fibroblasts. Scale bar: 1 mm. (G) Cell migration or invasion index was calculated as the number of cells attached to the bottom of control or Matrigel-coated membranes after 24 hours, normalized to respective Si-CTL lung fibroblasts (n = 3 per group). Throughout, data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.01 by 1-way ANOVA (A) or Student’s t test (G).

Article Snippet: For CD274-KO, we first generated a Cas9-expressing cell line (Invitrogen LentiArray Cas9 Lentivirus, {"type":"entrez-nucleotide","attrs":{"text":"A32069","term_id":"1249524"}} A32069 , Thermo Fisher Scientific), and then sgRNA expression clones targeting CD274 (HCP208443-SG01-3-10, GeneCopoeia) and scrambled sgRNA control plasmid (CCPCTR01-SG01-10, GeneCopoeia) were used to generate CD274-KO and control cells.

Techniques: Gene Expression, Western Blot, Expressing, In Vitro, Migration, Invasion Assay, Control

Journal: JCI Insight

Article Title: PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis

doi: 10.1172/jci.insight.125326

Figure Lengend Snippet: (A–C) Equal numbers of cells were seeded in the upper part of transwells and cell migration and invasion assays were performed (n = 3 per group). (A) Representative images of migrating and invasive CD274– and CD274hi IPF fibroblasts treated with VS4718 or vehicle (DMSO). Scale bar: 1 mm. (B and C) Cell migration or invasion index was calculated as the number of cells attached to the bottom of control or Matrigel-coated membranes after 24 hours, normalized to respective CD274– lung fibroblasts. (D) Western blot analyses of CD274, PDCD1LG2, p-FAK1, and FAK1. GAPDH served as loading control. See complete unedited blots in the supplemental material. Neg, negative. Scale bars: 1 mm. Throughout, data are the mean ± SEM. *P < 0.05; **P < 0.01 by 1-way ANOVA (B and C).

Article Snippet: For CD274-KO, we first generated a Cas9-expressing cell line (Invitrogen LentiArray Cas9 Lentivirus, {"type":"entrez-nucleotide","attrs":{"text":"A32069","term_id":"1249524"}} A32069 , Thermo Fisher Scientific), and then sgRNA expression clones targeting CD274 (HCP208443-SG01-3-10, GeneCopoeia) and scrambled sgRNA control plasmid (CCPCTR01-SG01-10, GeneCopoeia) were used to generate CD274-KO and control cells.

Techniques: Migration, Control, Western Blot

Journal: JCI Insight

Article Title: PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis

doi: 10.1172/jci.insight.125326

Figure Lengend Snippet: Masson’s trichrome staining of collagen in lung sections (A and B) and hydroxyproline content in lung tissues (C and D) from NSG mice injected with CD274– and CD274hi IPF fibroblasts treated with VS4718, vehicle control CMC-Na, or from NSG mice that received gRNA control (gRNA-CTL) or CD274-KO lung fibroblasts (n = 6 per group). Neg, negative. Scale bars (A and B): 1 mm (top panel), 100 μm (middle and lower panels). Throughout, data are the mean ± SEM. *P < 0.05 by 1-way ANOVA (C) or Student’s t test (D).

Article Snippet: For CD274-KO, we first generated a Cas9-expressing cell line (Invitrogen LentiArray Cas9 Lentivirus, {"type":"entrez-nucleotide","attrs":{"text":"A32069","term_id":"1249524"}} A32069 , Thermo Fisher Scientific), and then sgRNA expression clones targeting CD274 (HCP208443-SG01-3-10, GeneCopoeia) and scrambled sgRNA control plasmid (CCPCTR01-SG01-10, GeneCopoeia) were used to generate CD274-KO and control cells.

Techniques: Staining, Injection, Control

Journal: JCI Insight

Article Title: PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis

doi: 10.1172/jci.insight.125326

Figure Lengend Snippet: Masson’s trichrome staining of collagen in lung sections (A) and hydroxyproline content in lung tissues (B) from NSG mice injected with CD274hi IPF fibroblasts treated with anti-CD274 (α-CD274) antibody (n = 12 per group) or isotype control IgG (n = 12 for days 0–35 IgG, n = 11 for days 35–63 IgG) on day 63 after fibroblast injection. Scale bars: 1 mm (top panel), 100 μm (middle and lower panels). Data are the mean ± SEM. *P < 0.05 by 2-way ANOVA (B).

Article Snippet: For CD274-KO, we first generated a Cas9-expressing cell line (Invitrogen LentiArray Cas9 Lentivirus, {"type":"entrez-nucleotide","attrs":{"text":"A32069","term_id":"1249524"}} A32069 , Thermo Fisher Scientific), and then sgRNA expression clones targeting CD274 (HCP208443-SG01-3-10, GeneCopoeia) and scrambled sgRNA control plasmid (CCPCTR01-SG01-10, GeneCopoeia) were used to generate CD274-KO and control cells.

Techniques: Staining, Injection, Control