Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Expression of markers of metabolic compartmentalization in non-small cell lung cancer (NSCLC). Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patient samples were stained for MCT4, MCT1 and TOMM20 by immunohistochemistry. For each marker, representative images were taken from tumor tissue and adjacent normal lung tissue within the same sample. (A, B) MCT4 expression in the tumor stroma of LUAD (A) and LUSC (B) and their corresponding adjacent normal lung. (C, D) MCT1 expression in carcinoma cells in LUAD (C) and LUSC (D) and their corresponding adjacent normal lung. (E, F) TOMM20 expression in carcinoma cells in LUAD (E) and LUSC (F) and their corresponding adjacent normal lung. Images were taken at 20X. (S, stroma; C, carcinoma cells).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Expressing, Staining, Immunohistochemistry, Marker

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Expression of markers of metabolic compartmentalization in co-cultures of ADT carcinoma cells and fibroblasts. Human ADT carcinoma cell lines A549, HCC827, H226, H23, SCC9 and SCC25 were co-cultured with BJ1 fibroblasts for 4 days. Monocultures of carcinoma cells and BJ1 fibroblasts were maintained in parallel as controls. (A–D) Confocal imaging of MCT4 in monocultures of BJ1 and in co-cultures of BJ1 with the NSCLC cells HCC827, H226 and H23 (A) and HNSCC cells SCC9 and SCC25 (C) , and their respective quantification of MCT4 staining in BJ1 (B, D) . (E, F) . Confocal imaging of IDH3α in monocultures of BJ1 and in co-cultures of BJ1 with A549 and HCC827 (E) , and quantification of IDH3α staining in BJ1 (F) . (G–L) Confocal imaging of TOMM20 in monocultures and co-cultures of A549 (G) , HCC827 (I) , and SCC9 (K) with BJ1, and quantification of TOMM20 staining in A549 (H) , HCC827 (J) and SCC9 (L) . Confocal microscopy images were acquired at the 40X magnification, with 1.5x zoom in panel (E) . For all images, MCT4, IDH3α and TOMM20 staining are shown in red, carcinoma cells are shown in green (K8/18 staining), and nuclei are shown in blue (DAPI). For all markers, red staining was quantified using ImageJ. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units. Scale bar = 50 μm).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Expressing, Cell Culture, Imaging, Staining, Confocal Microscopy

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: PEPCK-M expression in glycolytic fibroblasts. BJ1 and WT or MCT4-KO MEF were co-cultured with ADT carcinoma cells. Monocultures of BJ1 and MEF were maintained in parallel as controls. (A–D) Confocal imaging of PEPCK-M in monocultures and in co-cultures of BJ1 with A549 (A) and SCC25 (C) , and their respective quantification of PEPCK-M staining in BJ1 (B, D) . (E, F) Confocal imaging of PEPCK-M in monocultures of WT and MCT4-KO MEF (E) and quantification of PEPCK-M staining (F) . (G–L) Confocal imaging of PEPCK-M in WT and MCT4-KO MEF in co-culture with A549 (G) , SCC9 (I) and SCC25 (K) , and their respective quantification of PEPCK-M staining in MEF (H, J, L) . Confocal microscopy images were acquired at the 40X magnification. For all images, PEPCK-M staining is shown in red, carcinoma cells are shown in green (K8/18 staining), and nuclei are shown in blue (DAPI). PEPCK-M staining was quantified using ImageJ. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units. Scale bar = 50 μm).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Expressing, Cell Culture, Imaging, Staining, Co-Culture Assay, Confocal Microscopy

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Effects of HIF-1α inhibition on fibroblast metabolism and carcinoma cell aggressiveness. Co-cultures of ADT carcinoma cells and BJ1 were treated with the HIF-1α inhibitor BAY 87-2243 or vehicle control for 48 hours. (A–D) Confocal imaging of IDH3α in co-cultures of BJ1 with A549 (A) and HCC827 (C) treated or untreated with BAY 87-2243, and their respective quantification of IDH3α staining in BJ1 (B, D) . (E–H) Confocal imaging of MCT4 in co-cultures of BJ1 with A549 (E) and HCC827 (G) treated or untreated with BAY 87-2243, and their respective quantification of MCT4 staining in BJ1 fibroblasts (F, H) . Confocal microscopy images were acquired at the 40X magnification, with 1.5x zoom in panels (A, C) . For all images, IDH3α and MCT4 staining are shown in red, carcinoma cells are shown in green (K8/18 staining), and nuclei are shown in blue (DAPI). IDH3α and MCT4 staining were quantified using ImageJ. (I–L) Flow cytometry assessment of percentage of carcinoma cells undergoing apoptosis (AnnV staining) and cell death (PI staining) in co-cultured A549 (I, J) and HCC827 (K, L) untreated or treated with BAY 87-2243 with representative flow cytometry plots showing gating strategy for AnnV (APC) and PI (PE) staining. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units. Scale bar = 50 μm).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Inhibition, Control, Imaging, Staining, Confocal Microscopy, Flow Cytometry, Cell Culture

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Effects of ROS neutralization and MCT4 downregulation on fibroblast metabolism and ADT carcinoma cell aggressiveness. Co-cultures of ADT carcinoma cells with BJ1 were treated with N-acetyl cysteine (NAC) for 24 hours for proliferation assessments, and for 48 hours for confocal imaging and apoptosis assessment. (A–D) Confocal imaging of MCT4 in co-cultures of BJ1 with A549 (A) and HCC827 (C) treated or untreated with NAC, and respective quantification of MCT4 staining in BJ1 (B, D) . Confocal microscopy images were acquired at the 40X magnification. For all images, MCT4 staining is shown in red, carcinoma cells are shown in green (K8/18 staining), and nuclei are shown in blue (DAPI). MCT4 staining was quantified using ImageJ. (E, F) Flow cytometry quantification of percentage of A549 cells in the DNA synthesis phase, measured by 5-ethynyl-2’-deoxyuridine (EdU) incorporation (E) , and gating strategy for EdU (Pacific Blue) and FxCycle (APC) staining (F) . (G–J) Flow cytometry assessment of percentage of carcinoma cells undergoing apoptosis (AnnV staining) and cell death (PI staining) in co-cultured A549 (G, H) and HCC827 (I, J) cells untreated or treated with NAC with representative flow cytometry plots showing gating strategy for AnnV (APC) and PI (PE) staining. (K–M) ADT carcinoma cells were co-cultured with mCherry-tagged BJ1 with downregulated MCT4 expression (BJ1-sgMCT4) or their control counterparts (BJ1-sgCTRL) and apoptosis (AnnV staining) and cell death (DAPI staining) was assessed in A549 (K) , HCC827 (L) and H520 (M) by flow cytometry. (N) GFP-tagged A549 were monocultured or co-cultured with WT or MCT4-KO MEFs and proliferation rates in A549 were assessed by flow cytometric analysis of EdU incorporation. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units. Scale bar = 50 μm). ns, not significant.

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Neutralization, Imaging, Staining, Confocal Microscopy, Flow Cytometry, DNA Synthesis, Cell Culture, Expressing, Control

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Effects of fibroblast MCT4 on tumor growth. (A–J) Tumor xenografts were generated by co-injecting human ADT carcinoma cells with fibroblasts expressing or lacking MCT4 into nude mice. (A) Tumor volume and (B) tumor weight of HCC827 + BJ1-sgCTRL/sgMCT4 xenografts harvested at 2.5 weeks post-implantation. (C, D) Immunohistochemical assessment of mCherry (to detect BJ1) and MCT4 expression in HCC827 + BJ1-sgCTRL/sgMCT4 tumors, and image acquisition at 40x (C) and 60x (D) . In panel (D) the area of carcinoma cells, mouse stroma, and injected mCherry-expressing BJ1 (*) are marked. (E) Tumor volume and (F) tumor weight of HCC827 + WT/MCT4-KO MEF xenografts harvested at 4 weeks post-implantation. (G) Tumor volume and (H) tumor weight of H520 + BJ1-sgCTRL/sgMCT4 xenografts harvested at 2 months post-implantation. (I) Tumor volume and (J) tumor weight of SCC25 + BJ1-sgCTRL/sgMCT4 xenografts harvested at 6 weeks post-implantation. (K–N) Syngeneic tumors were generated by co-injecting C57BL/6 carcinoma cells with WT or MCT4-KO MEFs into WT or MCT4-KO C57BL/6 mice. (K) Tumor volume and (L) tumor weight of MOC1 + WT/MCT4-KO MEF tumors harvested at 7 weeks post-implantation. (M) Tumor volume and (N) tumor weight of MOC2 + WT/MCT4-KO MEF tumors harvested at 3 weeks post-implantation. Student’s t-test was used for statistical analyses (*p<0.05). (a.u., arbitrary units).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Generated, Expressing, Immunohistochemical staining, Injection

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Effects of fibroblast MCT4 on tumor metabolism. The metabolic markers MCT4, GLUT1, MCT1 and TOMM20 were assessed by immunohistochemical staining in tumor xenografts generated from the co-injection of HCC827 with BJ1-sgCTRL or BJ1-sgMCT4. Representative IHC images of MCT4 (A) , GLUT1 (D) , MCT1 (G) and TOMM20 (J) staining. Membranous staining of MCT4 (B, C) , GLUT1 (E, F) and MCT1 (H, I) in HCC827 cells was identified with the ImmunoMembrane software and quantified with ImageJ. The ImmunoMembrane software detects and labels strong membranous staining in red and weak staining in green. Only staining labeled in red was quantified by ImageJ as the area in pixels of the total image covered by the red labeling. Mitochondrial staining of TOMM20 on HCC827 cells was quantified by Aperio (K) . Representative images for each group are shown. All IHC images were acquired at 40X, except for MCT4 quantification in panel (B) that is 20X. Student’s t-test was used for statistical analyses (*p<0.05).

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Immunohistochemical staining, Staining, Generated, Injection, Software, Labeling

Journal: Frontiers in Oncology

Article Title: Monocarboxylate Transporter 4 in Cancer-Associated Fibroblasts Is a Driver of Aggressiveness in Aerodigestive Tract Cancers

doi: 10.3389/fonc.2022.906494

Figure Lengend Snippet: Metabolic compartmentalization in ADT cancers and interventions to modulate fibroblast metabolism and carcinoma cell aggressiveness. (A) We have demonstrated that both in patients and in experimental models of ADT cancers, CAFs are glycolytic, whereas carcinoma cells are mitochondria-rich and have OXPHOS metabolism. We have shown that CAFs have increased levels of ROS. ROS are a mean of communication between metabolic compartments and known inducers of HIF-1α stabilization in CAFs. HIF-1α drives glycolysis and transcriptionally regulates MCT4 and PEPCK-M. We have shown that CAFs have upregulation of MCT4 and PEPCK-M, and that PEPCK-M expression may also be regulated by MCT4. MCT4 is located in the plasma membrane and is the main exporter of glycolysis-derived lactate. Loss of IDH3α is also a driver of HIF-1α stabilization. We have shown that CAFs have downregulation of IDH3α expression and that elevated levels of HIF-1α may in turn maintain the low expression of IDH3α. Both upregulation of MCT4 and loss of IDH3α are markers of glycolysis in CAFs, and we define PEPCK-M as another possible marker of glycolytic reprogramming in CAFs. We have demonstrated that CAF glycolytic reprogramming can be targeted using a HIF-1α inhibitor (BAY 87-2243) and an antioxidant (NAC). In the carcinoma cell compartment, we have demonstrated that the presence of glycolytic CAFs induces ROS, MCT1 and TOMM20 expression and drives carcinoma cell aggressiveness, as seen by decreased cell death and increased proliferation, and tumor growth. In bold are the elements studied in our report. Black arrows indicate previously described pathways. Green arrows indicate the novel findings reported here. Red inhibitor arrows indicate NAC and BAY 87-2243 targets. (B) We have modulated CAF glycolytic metabolism and ADT carcinoma cell aggressiveness by means of pharmacological and genetic interventions. The HIF-1α inhibitor BAY 87-2243 rescued IDH3α expression in CAFs and induced carcinoma cell apoptosis. The antioxidant NAC reduced ROS levels and MCT4 expression in CAFs, and in carcinoma cells it reduced ROS levels, induced apoptosis and decreased proliferation. Genetic knock-down (KD) or knock-out (KO) of MCT4 in CAFs increased apoptosis and decreased proliferation in carcinoma cells, and reduced growth and the expression of MCT1, TOMM20, MCT4 and GLUT1 in tumors. Yellow boxes indicate effects in vitro . Red boxes indicate effects in vivo . CAF, Cancer-associated fibroblast; ROS, reactive oxygen species; HIF-1α, hypoxia inducible factor 1 alpha; MCT4, monocarboxylate transporter 4; IDH3α, isocitrate dehydrogenase 3 alpha; PEPCK-M, mitochondrial phosphoenolpyruvate carboxykinase; NAC, N-acetyl cysteine; MCT1, monocarboxylate transporter 1; TOMM20, translocase of the outer mitochondrial membrane 20. Created with BioRender.com .

Article Snippet: Sg RNA lentiviral expression clones targeting SLC16A3 (HCP253996-LvSG02), Sg RNA lentiviral control for pCRISPR-LvSG02 (CCPCTR01-LvSG02), and Cas9 nuclease lentiviral expression clone 1 (CP-LvC9NU-01) were purchased from GeneCopoeia.

Techniques: Expressing, Clinical Proteomics, Membrane, Derivative Assay, Marker, Knockdown, Knock-Out, In Vitro, In Vivo

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