Journal: Cancer Research

Article Title: Epstein–Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment

doi: 10.1158/0008-5472.can-20-3121

Figure Lengend Snippet: Figure 1. EBV infection in human adipocytes. A, Phenotypic changes of adipocytes (black arrows) at the invasive front of EBV-positive human salivary LELC tissue sections. Positive signals for EBER in in situ hybridization (brown). Scale bar, 100 mm. B, Detection of EaD expressed in adipocytes near tumor beds. IHC images of bone marrow tumor sections obtained from patients with NPC diagnosed with bone metastasis. Images were captured from tumor proximal (left and middle) and distal areas (right). Arrows, EaD-positive adipocytes. Scale bar, 50 mm. C, Invitro infection of human adipocyteswith EBV. Microscopic visualization of viral thymidinekinase_GFP (green) signals in live adipocytes at various days after EBV infection. Control adipocytes were treated with culture supernatant derived from EBV-negative AKATA cells. Scale bar, 50 mm.

Article Snippet: Antibodies used forWestern blotting, immunofluorescence, or IHC are as follows: phospho-p70S6K1 (T389; Cell Signaling Technology), p70S6K1 (Abcam), GAPDH (Abcam), p-HSL (Ser 563; Cell Signaling Technology), HSL (Cell Signaling Technology), EBV early antigen D (EaD; Abcam), EBV thymidine kinase (TK; LSBio), Rta (Argene), Zta (Santa Cruz Biotechnology), S100A4 (Cell Signaling Technology), phospho-AMPK (T172; Cell Signaling Technology), AMPK (Abcam), phospho-mTOR (S2448; Cell Signaling Technology), mTOR (Cell Signaling Technology), phospho-S6 (S240/244; Cell Signaling Technology), S6 (Cell Signaling Technology), ATGL (Abcam), MGL (Abcam,), phospho-ERK1/2 (T202/Y204), ERK1/2, phospho-GSK3a/b (S21/9), GSK-a/b, and FAP-a.

Techniques: Infection, In Situ Hybridization, Control, Derivative Assay

Journal: Cancer Research

Article Title: Epstein–Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment

doi: 10.1158/0008-5472.can-20-3121

Figure Lengend Snippet: Figure 2. Detection of EBV-encoded gene products in infected adipocytes. A–F, Assessment of mRNA expression of viral-encoded genes comprising EBNA1 (A), EBER1 (B), TK (C), Zta (D), LMP2A (E), and EaD (F) in adipocytes infected with EBV either derived from HK1EBV cells or rAKATA cells. Total RNA was extracted at specific time points after EBV infection as indicated. RNA extracted from B95.8 cellswere used as positive controls for EBV-encoded transcripts. mRNA expressionwas normalized to that of GAPDH. G, Detection of EBV episomal DNA in adipocytes infected with EBV derived from HK1EBV or rAKATA cells. DNA extracted from HK1EBV and B95.8 cells were used as positive controls for EBV episomes. Histograms showed quantification of normalized expression. Values are expressed as mean and SD of experiments performed in duplicate. H, Expression of EBV-encoded EaD (green, left), TK (red, middle), and LMP1 (green, right) in adipocytes infected with EBV from HK1EBV cells. Cells were fixed at 3 dpi. Nuclei were stained with Hoechst 33342. Scale bar, 20 mm. I, Western blot analysis of EBV-encoded TK, LMP1, and EaD expressions in adipocytes infected with EBV from rAKATA cells. Protein lysates were harvested at the indicated time points post infection. B-Actin was used as a loading control. Adipocytes treated with culture supernatants from EBV-negative AKATA or HK1 cells were used as uninfected controls.

Article Snippet: Antibodies used forWestern blotting, immunofluorescence, or IHC are as follows: phospho-p70S6K1 (T389; Cell Signaling Technology), p70S6K1 (Abcam), GAPDH (Abcam), p-HSL (Ser 563; Cell Signaling Technology), HSL (Cell Signaling Technology), EBV early antigen D (EaD; Abcam), EBV thymidine kinase (TK; LSBio), Rta (Argene), Zta (Santa Cruz Biotechnology), S100A4 (Cell Signaling Technology), phospho-AMPK (T172; Cell Signaling Technology), AMPK (Abcam), phospho-mTOR (S2448; Cell Signaling Technology), mTOR (Cell Signaling Technology), phospho-S6 (S240/244; Cell Signaling Technology), S6 (Cell Signaling Technology), ATGL (Abcam), MGL (Abcam,), phospho-ERK1/2 (T202/Y204), ERK1/2, phospho-GSK3a/b (S21/9), GSK-a/b, and FAP-a.

Techniques: Infection, Expressing, Derivative Assay, Staining, Western Blot, Control

Journal: Cancer Research

Article Title: Epstein–Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment

doi: 10.1158/0008-5472.can-20-3121

Figure Lengend Snippet: Figure 3. EBV infection induces lipolysis of adipocytes. A, Delipidation of EBV-infected adipocytes. LDs were visualized with BODIPY (green) in live adipocytes on day 4 after infection. Scale bar, 20 mm. B, Measurement of LD sizes based on BODIPY signals. Yellow indicates the LD areas used for quantification. Scale bar, 20 mm. C, Quantification of LD areas depicted in B. LD areas were calculated using CellSens imaging software (Olympus). Values are expressed as mean and SEM of at least three independent experiments. , P < 0.001, paired t test. D–G, Assessment of mRNA expression of key genes involved in lipid metabolism. Total RNA was extracted from uninfected adipocytes or adipocytes infected with EBV either derived from HK1EBV or rAKATA cells at the indicated time points after infection. Expression was normalized to that of GAPDH. Values are expressed as mean and SD of two independent experiments. , P < 0.05, paired t test. H, Western blot analysis of key molecules involved in lipid metabolism. Protein lysates were harvested at 24 hours post-EBV infection. GAPDH was used as a loading control. I, Increased expression of activated HSL [p-HSL(S563)] (red) in EBV-challenged adipocytes. Cells were fixed at 6 dpi. Nuclei were stained with Hoechst 33342. Scale bar, 20 mm. J, Expression of EBERs and p-HSL(S563) in adipocytes near tumor beds. Biopsy samples were obtained from patients diagnosed with bone marrow metastatic NPC. Scale bar, 50 mm. K, Increased FFA and glycerol levels released from EBV-challenged adipocytes. Culture supernatants were harvested at the indicated time points. Histogram showing quantification of normalized concentrations (mmol/L). Values are expressed as mean SD of experiments performed in duplicate. L, Kaplan–Meier recurrence-free and metastasis-free survival analysis of NPC patients based on serum FFA levels.

Article Snippet: Antibodies used forWestern blotting, immunofluorescence, or IHC are as follows: phospho-p70S6K1 (T389; Cell Signaling Technology), p70S6K1 (Abcam), GAPDH (Abcam), p-HSL (Ser 563; Cell Signaling Technology), HSL (Cell Signaling Technology), EBV early antigen D (EaD; Abcam), EBV thymidine kinase (TK; LSBio), Rta (Argene), Zta (Santa Cruz Biotechnology), S100A4 (Cell Signaling Technology), phospho-AMPK (T172; Cell Signaling Technology), AMPK (Abcam), phospho-mTOR (S2448; Cell Signaling Technology), mTOR (Cell Signaling Technology), phospho-S6 (S240/244; Cell Signaling Technology), S6 (Cell Signaling Technology), ATGL (Abcam), MGL (Abcam,), phospho-ERK1/2 (T202/Y204), ERK1/2, phospho-GSK3a/b (S21/9), GSK-a/b, and FAP-a.

Techniques: Infection, Imaging, Software, Expressing, Derivative Assay, Western Blot, Control, Staining

Journal: Cancer Research

Article Title: Epstein–Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment

doi: 10.1158/0008-5472.can-20-3121

Figure Lengend Snippet: Figure 5. EBV infection triggers dedifferentiation of adipo- cytes. A, Dedifferentiation processes of EBV- infected adipocytes. Image of fibroblasts isolated from nasopharyngeal biopsies was used as a phenotypic reference. Images were captured at the indicated time points. Scale bar, 50 mm. B, Representative images of fibroblast-like cellular structures (black arrows)in areas between adipose tissues and tumor beds. Brown, EaD. Left, bone marrow metastatic NPC section. Right, salivary LELC tissue sections. Scale bar, 50 mm. C, S100A4 protein signals (red) were increased in LMP1 (green) positive-dedifferentiated adipo- cytes. Images were obtained at 8 and 20 dpi. Adipocytes treated with supernatants from EBV-negative HK1 cells were used as uninfected control. Blue, DNA. Scale bar, 20 mm. D, IHC anal- ysis of EBERs, S100A4, and FAPa expression in paraffin-embedded consecutive bone marrow metastatic NPC tissue sections. Fibrotic response (blue) within tumor sections was evaluated using trichrome staining. Scale bar, 50 mm.

Article Snippet: Antibodies used forWestern blotting, immunofluorescence, or IHC are as follows: phospho-p70S6K1 (T389; Cell Signaling Technology), p70S6K1 (Abcam), GAPDH (Abcam), p-HSL (Ser 563; Cell Signaling Technology), HSL (Cell Signaling Technology), EBV early antigen D (EaD; Abcam), EBV thymidine kinase (TK; LSBio), Rta (Argene), Zta (Santa Cruz Biotechnology), S100A4 (Cell Signaling Technology), phospho-AMPK (T172; Cell Signaling Technology), AMPK (Abcam), phospho-mTOR (S2448; Cell Signaling Technology), mTOR (Cell Signaling Technology), phospho-S6 (S240/244; Cell Signaling Technology), S6 (Cell Signaling Technology), ATGL (Abcam), MGL (Abcam,), phospho-ERK1/2 (T202/Y204), ERK1/2, phospho-GSK3a/b (S21/9), GSK-a/b, and FAP-a.

Techniques: Infection, Isolation, Control, Expressing, Staining

Journal: Cancer Cell

Article Title: Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity

doi: 10.1016/j.ccell.2020.04.013

Figure Lengend Snippet:

Article Snippet: Tritium thymidine (Methyl-3 H ) , PerkinElmer , Cat# NET027250UC.

Techniques: Recombinant, Lysis, Enzyme-linked Immunosorbent Assay, Luminex, Cell Isolation, Isolation, Transgenic Assay, Software