Journal: Cell Reports Medicine

Article Title: Human T Cells Expressing a CD19 CAR-T Receptor Provide Insights into Mechanisms of Human CD19-Positive β Cell Destruction

doi: 10.1016/j.xcrm.2020.100097

Figure Lengend Snippet:

Article Snippet: CAGGS-AsCpf1-2A-GFP-U6-sgRNA-cloning vector , This study , Addgene 159281.

Techniques: Recombinant, Cytotoxicity Assay, Purification, Luminex, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Labeling, Plasmid Preparation, Software

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: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Development of galectin-3 knockdown ARPE-19 cells. ( A , B ) Merged phase-contrast and fluorescent imaging for GFP expression of ARPE-19 cells without ( A ) and with pCRISPR-LGALS3 transfection ( B ). ( C , D ) Immunostaining for galectin-3 of ARPE-19 ( C ) and ARPE-19/LGLAS3 +/− cells ( D ) after FACS and single-cell cultivation. ( E , F ) Phase-contrast image of ARPE-19 ( E ) and ARPE-19/LGLAS3 +/− cells ( F ). Magnification bar in ( A , B ) 100 µm; in ( C , D ) 20 µm, in ( E , F ) 100 µm; blue, Hoechst staining. ( G ) Western blot analysis for galectin-3 of ARPE-19 and ARPE-19/LGLAS3 +/− cells. ( H , I ) Galectin-3 concentration of conditioned cell culture medium ( H ) and cell lysate ( I ) from ARPE-19 and ARPE-19/LGLAS3 +/− cells after incubation in unsupplemented cell culture medium for 72 h. Mean ± SD; ** p < 0.01; **** p < 0.0001; n = 10.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Knockdown, Imaging, Expressing, Transfection, Immunostaining, Staining, Western Blot, Concentration Assay, Cell Culture, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Reduced expression of galectin-3 decreases viability of immortalized RPE cells in vitro. WST-1 assay of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation in cell culture medium without supplementation and various concentrations of hr-galectin-3 (hr-Gal3) for 72 h. Mean ± SD; **** p < 0.0001; n ≥ 28 of at least 4 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, In Vitro, WST-1 Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Decreased expression of galectin-3 declines proliferation of immortalized RPE cells in vitro. BrdU ELISA of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation in cell culture medium without supplementation for 72 h with and without various concentrations of hr-galectin-3 ( A ) and/or an additional treatment with 100mM lactose ( B ). Mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n ≥ 20 of at least 4 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Decreased galectin-3 expression reduces migration of ARPE-19 cells in vitro. Quantification ( A ) and representative images ( B ) of scratch migration assay of native ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation with and without hr-galectin-3 in cell culture medium without supplements for 24 h. ( A ) For quantification, the recolonized area after 24 h was calculated and plotted as the relative recolonized area. ( B ) Representative images of native ARPE-19 (left panel) and ARPE-19/LGALS3 +/− cells (right panel) immediately after scratching (0 h, upper row) and after incubation for 24 h (lower panel). Mean ± SD; *** p < 0.001; **** p < 0.0001; n ≥ 10 of 3 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Migration, In Vitro, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Reduced expression of endogenous galectin-3 enhances cell attachment of immortalized RPE cells in vitro. ( A ) Cell adhesion of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells was quantified 30, 60, 90, 120, 150, and 180 min after seeding and plotted as the relative number of adherent cells. Mean ± SD; n = 12 for each group of 3 independent experiments; * comparison of ARPE-19 versus ARPE-19/LGALS3 +/− cells; # comparison of ARPE-19/FACS versus ARPE-19/LGALS3 +/− cells; **** p < 0.001; #### p < 0.001. ( B ) Relative number of adherent ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells 90 min after seeding and incubation with various concentrations of hr-galectin-3, which was added immediately before seeding. Mean ± SD; n = 15 for each group of 3 independent experiments; ** p < 0.01; **** p < 0.0001.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Cell Attachment Assay, In Vitro, Comparison, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Lack of endogenous galectin-3 promotes epithelial-to-mesenchymal transition of immortalized RPE. ( A – F ) Phaloidin staining (( A , B ) orange) as well as immunofluorescent staining for sm-α-actin (( C , D ) orange) and N-cadherin ( E , F ) green) of ARPE-19 ( A , C , E ) and ARPE-19/LGALS3 +/− cells ( B , D , F ). Scale bar, 20 µm; blue, DAPI staining. ( G ) Real-time rt-PCR for sm-α-actin, E-cadherin, and N-cadherin mRNA expression of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells. Mean ± SD; n ≥ 7 of 5 independent experiments; * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Staining, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Endogenous galectin-3 expression maintains basal pAKT, pERK, and β-catenin signaling in immortalized RPE cells. Western blot analysis ( A ) and densitometry for pAKT ( B ), pERK ( C ), and β-catenin ( D ) expression of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells. Mean ± SD; n ≥ 6 of 6 independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Western Blot

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: 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

Journal: Molecular Cancer

Article Title: The PI3K subunits, P110α and P110β are potential targets for overcoming P-gp and BCRP-mediated MDR in cancer

doi: 10.1186/s12943-019-1112-1

Figure Lengend Snippet: Reversal of MDR ability via knockout of target P110 subunits, including P110α (PIK3CA) and P110β (PIK3CB), from MDR cancer cell populations of KB-C2 and H460/MX80 with pCRISPR-CG01 all-in-one plasmid. a Map of pCRISPR-CG01 . b Analysis with Western blot confirming the knockout of PIK3CA and PIK3CB that is of low abundance in KB-C2 and H460/MX80, as compared with GAPDH. To correct possible result deviation caused by exposure saturation of partial bands, less exposure of GAPDH bands was used to indicate relative cell counts. The absence or intensity-reduction of the target bands are depicted by red stars. Truncated proteins are depicted by head-down arrows. Relative quantification was carried out with ImageQuant TL based on the intensity of the bands. Statistical calculation was made based on three independent repeats. c PCR as adjuvant method to characterize target P110 subunit deficiency in KB-C2 and H460/MX80 cells. Missing bands or reduced copies of the target PCR products are depicted by red stars. New bands generated by chromosomal recombination are depicted by diamonds. Truncated fragments are depicted by head-down arrows. d - f MTT assay showing changes in MDR level of the KB-C2 and H460/MX80 cells with target PI3K 110α or 110β subunits knocked out. Colchicine and paclitaxel, the substrates of P-gp, were used for evaluation of reversal of KB-C2 over-expressing P-gp. The BCRP substrate mitoxantrone was used for analysis of reversal of MDR of H460/MX80 with BCRP over-expressed. The experiments were performed at least three times

Article Snippet: To avoid the potential risk of un-specified chromosomal recombination during transfection mediated by viral vectors, polymer-based GenePORTER transfection reagents for gene delivery were mixed with plasmids HCP213150-CG01–1-10 and HCP213151-CG01–1-10, developed as sgRNA/Cas9 all-in-one expression clones respectively targeting PIK3CA (NM_006218.2) and PIK3CB (NM_006219.1) (GeneCopoeia Inc., Rockville, MD).

Techniques: Knock-Out, Plasmid Preparation, Western Blot, Quantitative Proteomics, Adjuvant, Generated, MTT Assay, Expressing