Journal: PLOS Pathogens

Article Title: The impact of fatty acid synthase on HSV-1 infection dynamics

doi: 10.1371/journal.ppat.1013068

Figure Lengend Snippet: (A) Western blot analysis of protein lysates from mock or infected cells at the indicated hpi using antibodies against ICP27, gD, or actin. A representative blot and the densitometric analyses are shown. Values were normalized to actin and plotted as fold induction relative to mock-infected cells (set at 1). ( n = 2; unpaired t-test). (B) At 24 hpi, the number of intracellular HSV-1 genomes was measured by qPCR ( n = 3; unpaired t-test). (C) At 48 hpi, the number of genome-containing particles released by cells was measured by qPCR ( n = 3; unpaired t-test). (D) The supernatants from panel C were used to determine the number of infectious units/mL by the viral yield test ( n = 3; unpaired t-test). (E) The genome-to-infectious unit ratio has been determined. Data are expressed as fold induction relative to shCTRL cells (set at 1) ( n = 3; unpaired t-test). (F) Surface CD36 protein expression in shCTRL and shFASN cells was assessed by flow cytometry. Results show the mean fluorescence intensity (MFI) ( n = 3; unpaired t-test). (G) SSO toxicity was determined at 48 h post-treatment (hpt), using the MTT method ( n = 3). (H) Schematic representation of FA uptake using a fluorescent analog of palmitate (BODIPY FL C16) (Created in BioRender. De Andrea, M. (2025) https://BioRender.com/j23i568 ). (I) shCTRL and shFASN cells were infected with HSV-1 in serum-free conditions and at 48 hpi treated with DMSO or SSO (200 µM) for 1 h. Then, cells were cultured in the presence of 2 μM BODIPY FL C16 for 30 minutes and then assessed by flow cytometry. Results show the MFI ( n = 3; one-way ANOVA followed by Bonferroni’s post-tests). (J) shCTRL and shFASN cells were treated with DMSO or SSO in the presence of serum and then infected with HSV-1 (MOI 1). At 48 hpi, the supernatants were used to determine the number of infectious units/mL by the viral yield test ( n = 3; unpaired t-test). Data are shown as the mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CMS121 (HY-135981, MedChemExpress), C75 (HY-12364, MedChemExpress), SSO (11211, Cayman Chemical), palmitate (P0500; Sigma-Aldrich), and BODIPY FL C16 (D3821; Invitrogen) were used at a concentration of 5 μM, 5 μM, 200 μM, 200 μM, and 2 μM, respectively. pLKO.1 puro-humanU6-shRNA FASN was a gift from Elizabeth Stoll (Addgene plasmid #82327; http://n2t.net/addgene:82327 ; RRID: Addgene_82327). pLKO.1 Puro shRNA Scramble was a gift from Antonia Follenzi (University of Piemonte Orientale, Novara, Italy). pAcUW51-CgE was a gift from Pamela Bjorkman (Addgene plasmid #13762; http://n2t.net/addgene:13762 ; RRID: Addgene_13762) [ ].

Techniques: Western Blot, Infection, Expressing, Flow Cytometry, Fluorescence, Cell Culture

Journal: iScience

Article Title: Metabolic modeling of single bronchoalveolar macrophages reveals regulators of hyperinflammation in COVID-19

doi: 10.1016/j.isci.2022.105319

Figure Lengend Snippet: The PPARγ agonist rosiglitazone reduces hyperinflammation in macrophages stimulated with SARS-CoV-2 or TLR7/8 agonist (A) GSEA analysis of long-chain fatty acid transport and lipid metabolic process gene sets in macrophages from severe versus mild COVID-19 patients. (B) Human primary monocyte-derived macrophages were stimulated with SARS-CoV-2 virus (MOI = 0.1) for 24 h. mRNA levels of CD36 , FABP4 , and ACAA2 were detected by real-time PCR. (C) GSEA analysis of PPAR signaling pathway in macrophages from severe versus mild COVID-19 patients. (D) Pearson correlation plot of metabolic scores versus PPAR pathway score in macrophages from BALF of HCs and patients with COVID-19. (E) Expression levels of PPARG in macrophages from BALF of patients with HC, mild or COVID-19. (F) Human primary monocyte-derived macrophages were stimulated with SARS-CoV-2 virus (MOI = 0.1) for 24 h. mRNA level of PPARG were detected by real-time PCR. Shown (B&F) are representative data (mock, n = 5; SARS-CoV-2, n = 5) from 3 independent donors with mean values. (G and H) Human primary macrophages were cultured in the presence of PPARγ inhibitor GW9662 (2μM). At 48 h, the cells were stimulated with R848. IL-6, TNF-α, and CCL2 levels in culture supernatant 24 h after stimulation. Shown are representative data (mock, n = 6; R848, n = 6, Rosi + R848, n = 6) from 3 independent donors with mean values. (I) Human primary macrophages were cultured in the presence of PPARγ activator rosiglitazone. At 48 h, the cells were incubated in medium containing 0.2μM BODIPY FL C16 for 20 min or in medium containing 0.1mg/mL pHrodo Green E. coli BioParticles Conjugate for 2 h at 37°C. (J–L) Uptake of fatty acids (J-K) and E. coli bioparticles (L) were compared between groups using flow cytometry. Shown are representative data from 2 independent donors with mean values. (M and N) Human primary macrophages were cultured in the presence of PPARγ activator rosiglitazone (5μM). At 48 h, the cells were stimulated with R848. IL-6, TNF-α, and CCL2 levels in culture supernatant 24 h after stimulation (N). Shown are representative data (mock, n = 6; R848, n = 6, Rosi + R848, n = 6) from 3 independent donors with mean values. (O) Human primary macrophages were cultured as in K, CD36 and FABP4 mRNA levels in cells were detected by real-time PCR 24 h after stimulation. (P–R) Human primary monocytes were differentiated to macrophages with M-CSF (colony stimulating factor 1) for 4 days in the presence of rosiglitazone. The differentiated cells were then stimulated with SARS-CoV-2 (MOI = 0.1) for 24 h (P). IL-6 and TNF-α levels in culture supernatant were detected by ELISA (Q). mRNA levels of CD36 and FABP4 in cells were detected by real-time PCR 24 h after stimulation (R). Shown are representative data (mock, n = 5; Rosi, n = 5, SARS-CoV-2, n = 5, Rosi + SARS-CoV-2, n = 5) from 3 independent donors with mean values. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Unpaired, two-tailed Student’s t test (for B, F, and L) or one-way ANOVA and Bonferroni’s post hoc test (for H, K, N, O, Q, and R) were performed to compare between groups.

Article Snippet: For measuring uptake of fatty acids, macrophages were incubated in medium containing 0.2μM BODIPY FL C16 (ThermoFisher, D3821) for 20minat 37°C.

Techniques: Derivative Assay, Virus, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Incubation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: Metabolic modeling of single bronchoalveolar macrophages reveals regulators of hyperinflammation in COVID-19

doi: 10.1016/j.isci.2022.105319

Figure Lengend Snippet:

Article Snippet: For measuring uptake of fatty acids, macrophages were incubated in medium containing 0.2μM BODIPY FL C16 (ThermoFisher, D3821) for 20minat 37°C.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Software, Activity Assay

Journal: bioRxiv

Article Title: Cancer-associated adipocytes mediate CD8 + T cell dysfunction via FGF21-driven lipolysis

doi: 10.1101/2024.10.18.618991

Figure Lengend Snippet: A-B. FACS analysis of neutral lipid content (BODIPY-493), fatty acid uptake (BODIPY-C16) and lipid peroxidation (BODIPY-C11) in CD8 + T cells treated with conditioned media from WT-AAD or tumor cell (E0771 or MC38)-educated adipocytes (Transwell-CAA) (A) , or E0771/MC38 tumor-adjacent CAAs (B) after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). C-D. FACS analysis of the percent of PD-1 hi , LAG-3 + , TIM-3 + of CD8 + T cells treated with conditioned media from WT-AAD or tumor cell (E0771 or MC38)-educated adipocytes (Transwell-CAA) (C) , or E0771/MC38 tumor-adjacent CAAs (D) after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). E-F. FACS analysis of the percent of IFNγ + , Granzyme B + , TNFα + and Perforin + of CD8 + T cells treated with conditioned media from WT-AAD or tumor cell (E0771 or MC38)-educated adipocytes (Transwell-CAA) (E) , or E0771/MC38 tumor-adjacent CAAs (F) after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). Data are presented as the mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 by Student’s t test (A, B) and two-way ANOVA (C, D, E, F).

Article Snippet: For the intracellular staining of lipid contents in T cells, cells were washed with PBS and fixed with fixation and permeabilization buffer (cat#88-8823-88, eBioscience) for 30 min. After that, cells were stained with 2 μM BODIPY-493/508 (cat#D3922, Invitrogen), 2 μM BODIPY-FL-C16 (cat#D3821, Invitrogen) or 2 μM BODIPY-581/591-C11 (cat#D3861, Invitrogen) for 30 min at 37°C in the dark.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Cancer-associated adipocytes mediate CD8 + T cell dysfunction via FGF21-driven lipolysis

doi: 10.1101/2024.10.18.618991

Figure Lengend Snippet: A. ELISA analysis of FGF21 protein levels in the supernatant from WT-AAD and E0771/MC38 tumor-adjacent CAAs (n = 3 biological replicates). B. Tumor growth curve of E0771 and MC38 in Fgf21 fl/fl and Fgf21 ΔAD mice (n = 5 mice). C. FACS analysis of the percent of tumor-infiltrating CD4 + and CD8 + T cells in E0771 and MC38 tumors from Fgf21 fl/fl and Fgf21 ΔAD mice (n =4-5 mice). D. GSEA analysis of gene signatures of lipid metabolism, fatty acid oxidation, CD8 + T cell exhaustion and function in CD8 + TILs sorted from MC38 tumors in Fgf21 fl/fl and Fgf21 ΔAD mice (n = 3 mice). E. FACS analysis of BODIPY-493, BODIPY-C16 and BODIPY-C11 staining in CD8 + TILs of E0771 and MC38 tumors in Fgf21 fl/fl and Fgf21 ΔAD mice (n = 4-5 mice). F. FACS analysis of PD-1, LAG-3 and TIM-3 in CD8 + TILs of E0771 and MC38 tumors in Fgf21 fl/fl and Fgf21 ΔAD mice (n = 4-5 mice). G. FACS analysis of IFNγ, Granzyme B, and Perforin in CD8 + TILs of E0771 and MC38 tumor in Fgf21 fl/fl and Fgf21 ΔAD mice (n = 4-5 mice). Data are presented as the mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001 by Student’s t test (A) and two-way ANOVA (B, C, E, F, G).

Article Snippet: For the intracellular staining of lipid contents in T cells, cells were washed with PBS and fixed with fixation and permeabilization buffer (cat#88-8823-88, eBioscience) for 30 min. After that, cells were stained with 2 μM BODIPY-493/508 (cat#D3922, Invitrogen), 2 μM BODIPY-FL-C16 (cat#D3821, Invitrogen) or 2 μM BODIPY-581/591-C11 (cat#D3861, Invitrogen) for 30 min at 37°C in the dark.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Cancer-associated adipocytes mediate CD8 + T cell dysfunction via FGF21-driven lipolysis

doi: 10.1101/2024.10.18.618991

Figure Lengend Snippet: A. Oil Red O staining of WT-AAD or E0771/MC38 tumor-adjacent CAAs from Fgf21 fl/fl and Fgf21 ΔAD mice (n = 3 biological replicates). B. FACS analysis of BODIPY-493 staining in WT-AAD or E0771/MC38 tumor-adjacent CAAs from Fgf21 fl/fl and Fgf21 ΔAD mice (n = 3 biological replicates). C. FACS analysis of BODIPY-493, BODIPY-C16 and BODIPY-C11 staining in CD8 + T cells treated with conditioned media from WT-AAD or E0771/MC38 tumor-adjacent CAAs from Fgf21 fl/fl and Fgf21 ΔAD mice after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). D. FACS analysis of the percent of PD-1 hi , LAG-3 + and TIM-3 + of CD8 + T cells treated with conditioned media from WT-AAD or E0771/MC38 tumor-adjacent CAAs from Fgf21 fl/fl and Fgf21 ΔAD mice after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). E. FACS analysis of the percent of IFNγ + , Granzyme B + , TNFα + and Perforin + of CD8 + T cells treated with conditioned media from WT-AAD or E0771/MC38 tumor-adjacent CAAs from Fgf21 fl/fl and Fgf21 ΔAD mice after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). Data are presented as the mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 by Student’s t test (A, B, C) and two-way ANOVA (D, E).

Article Snippet: For the intracellular staining of lipid contents in T cells, cells were washed with PBS and fixed with fixation and permeabilization buffer (cat#88-8823-88, eBioscience) for 30 min. After that, cells were stained with 2 μM BODIPY-493/508 (cat#D3922, Invitrogen), 2 μM BODIPY-FL-C16 (cat#D3821, Invitrogen) or 2 μM BODIPY-581/591-C11 (cat#D3861, Invitrogen) for 30 min at 37°C in the dark.

Techniques: Staining, Activation Assay

Journal: bioRxiv

Article Title: Cancer-associated adipocytes mediate CD8 + T cell dysfunction via FGF21-driven lipolysis

doi: 10.1101/2024.10.18.618991

Figure Lengend Snippet: A. GSEA analysis of gene signatures of lipid lipolysis in MC38 tumor-adjacent adipose tissues from Fgf21 fl/fl and Fgf21 ΔAD mice (n = 3 mice). B. qPCR analysis of gene expression of lipolytic lipases in WT normal adipose tissues and E0771/MC38 tumor-adjacent adipose tissues from Fgf21 fl/fl and Fgf21 ΔAD mice (n = 3 mice). C. Western Blot analysis of ATGL expression in WT normal adipose tissues and E0771 tumor-adjacent adipose tissues from Fgf21 fl/fl and Fgf21 ΔAD mice (n = 3 independent experiments). D. Western Blot analysis of p-FGFR1/FGFR1, KLB, p-p38/p38 in WT-AAD, and Fgf21 fl/fl and Fgf21 ΔAD transwell CAAs educated by E0771/MC38 tumor cells for 6 hr (n = 3 independent experiments). E. qPCR analysis of Atgl expression in WT-AAD, E0771/MC38 tumor cell-educated CAAs, plus with P38 inhibitor and FGFR1 inhibitor treatment. F. FACS analysis of BODIPY-493, BODIPY-C16 and BODIPY-C11 staining in CD8 + T cells treated with conditioned media from WT-AAD or E0771/MC38 tumor cell-educated CAAs, plus with ATGL inhibitor treatment, after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). G. FACS analysis of the percent of PD-1 hi , LAG-3 + and TIM-3 + of CD8 + T cells treated with conditioned media from WT-AAD or E0771/MC38 tumor cell-educated CAAs, plus with ATGL inhibitor treatment, after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). H. FACS analysis of the percent of IFNγ + , Granzyme B + , TNFα + and Perforin + of CD8 + T cells treated with conditioned media from WT-AAD or E0771/MC38 tumor cell-educated CAAs, plus with ATGL inhibitor treatment, after activation by anti-CD3/CD28 for 96 hr (n = 3 biological replicates). Data are presented as the mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 by Student’s t test (E) and two-way ANOVA (B, F, G, H).

Article Snippet: For the intracellular staining of lipid contents in T cells, cells were washed with PBS and fixed with fixation and permeabilization buffer (cat#88-8823-88, eBioscience) for 30 min. After that, cells were stained with 2 μM BODIPY-493/508 (cat#D3922, Invitrogen), 2 μM BODIPY-FL-C16 (cat#D3821, Invitrogen) or 2 μM BODIPY-581/591-C11 (cat#D3861, Invitrogen) for 30 min at 37°C in the dark.

Techniques: Expressing, Western Blot, Staining, Activation Assay

Journal: bioRxiv

Article Title: Cancer-associated adipocytes mediate CD8 + T cell dysfunction via FGF21-driven lipolysis

doi: 10.1101/2024.10.18.618991

Figure Lengend Snippet: A. Tumor growth curve of E0771 and MC38 tumors treated with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1 (n = 5 mice). B. FACS analysis of the percent of tumor-infiltrated CD4 + , CD8 + T cells in E0771 and MC38 tumors after treatment with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1 (n = 5 mice). C. FACS analysis of BODIPY-493, BODIPY-C16 and BODIPY-C11 staining of CD8 + TILs in E0771 and MC38 tumors after treatment with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1 (n = 5 mice). D. FACS analysis of PD-1, LAG-3 and TIM-3 in CD8 + TILs in E0771 and MC38 tumors after treatment with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1 (n = 5 mice). E. FACS analysis of IFNγ and Granzyme B in CD8 + TILs in E0771 and MC38 tumors with Vehicle, anti-ATGL, anti-PD-1, or ATGL inhibitor combined with anti-PD-1 (n = 5 mice). F. IF staining of ATGL in tumor-adjacent and distant adipose tissues from breast cancer patients (n=18 patients). G. ELISA analysis of FGF21 protein levels in tumor-adjacent and distant adipose tissues from breast cancer patients (n=18 patients). H. Linear regression analysis of the correlation of FGF21 and ATGL expression in tumor-adjacent adipose tissues from breast cancer patients (n=18 patients). I. Linear regression analysis of the correlation of FGF21 levels in tumor-adjacent adipose tissues and the number of CD8 + PD-1 + cells or CD8 + Granzyme B + cells in tumor tissues (n=13 patients). J. Linear regression analysis of the correlation of ATGL expression in tumor-adjacent adipose tissue and the number of CD8 + PD-1 + cells or CD8 + Granzyme B + cells in tumor tissues (n=13 patients). Data are presented as the mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 by Student’s t test (F, G), two-way ANOVA (A, B, C, D, E) and liner regression analysis (H, I, J).

Article Snippet: For the intracellular staining of lipid contents in T cells, cells were washed with PBS and fixed with fixation and permeabilization buffer (cat#88-8823-88, eBioscience) for 30 min. After that, cells were stained with 2 μM BODIPY-493/508 (cat#D3922, Invitrogen), 2 μM BODIPY-FL-C16 (cat#D3821, Invitrogen) or 2 μM BODIPY-581/591-C11 (cat#D3861, Invitrogen) for 30 min at 37°C in the dark.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: bioRxiv

Article Title: Cancer-associated adipocytes mediate CD8 + T cell dysfunction via FGF21-driven lipolysis

doi: 10.1101/2024.10.18.618991

Figure Lengend Snippet: A. Images of tumor mass and tumor weight of E0771 and MC38 in the mice with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1 treatment (n = 5 mice). B. Representative FACS analysis of tumor-infiltrated CD4 + and CD8 + T cells in E0771 and MC38 tumors after treatment with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1. C. Representative FACS analysis of BODIPY-493, BODIPY-C16 and BODIPY-C11 staining in CD8 + TILs of E0771 and MC38 tumors after treatment with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1. D. Representative FACS analysis of PD-1, LAG-3 and TIM-3 staining in CD8 + TILs of E0771 and MC38 tumors after treatment with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1. E. Representative FACS analysis of IFNγ, Granzyme B staining of in CD8 + TILs of E0771 and MC38 tumors after treatment with Vehicle, ATGL inhibitor, anti-PD-1, or ATGL inhibitor combined with anti-PD-1. F. Representative IF staining of CD8 + , Granzyme B + and PD-1 + cells in tumor tissues of breast cancer patients. Scale bar: 50 μm. Data are presented as the mean±SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 by Student’s t test (A).

Article Snippet: For the intracellular staining of lipid contents in T cells, cells were washed with PBS and fixed with fixation and permeabilization buffer (cat#88-8823-88, eBioscience) for 30 min. After that, cells were stained with 2 μM BODIPY-493/508 (cat#D3922, Invitrogen), 2 μM BODIPY-FL-C16 (cat#D3821, Invitrogen) or 2 μM BODIPY-581/591-C11 (cat#D3861, Invitrogen) for 30 min at 37°C in the dark.

Techniques: Staining