Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti-T. Gondii Immunity and Tumor Immunotherapy.

doi: 10.1002/advs.202400952

Figure Lengend Snippet: Figure 5. Mice vaccinated with ME49Δompdc/gra4 activates stronger IFN-I responses and completely inhibit tumor growth. A) Body weight change of WT and Ifnar−/- mice vaccinated with ME49Δompdc or ME49Δompdc/gra4. B) qPCR analysis of ITS-1 gene expression in splenocytes from mice with or without ME49wt, ME49Δompdc, and ME49Δompdc/gra4 infection. C) qPCR analysis of Ifnb and Isg15 gene expression in splenocytes from mice with or without ME49Δompdc and ME49Δompdc/gra4 immunization. D) ELISA of IFN-𝛽production in serum from mice with or without ME49Δompdc and ME49Δompdc/gra4 immunization. E) Tumor growth (left) and survival curve (right) of WT mice vaccinated with ME49Δompdc, ME49Δompdc/gra4 or PBS, followed by implanted B16-F10 tumor cells. F) The size and location of the tumors detected in mice (top), and changes in the tumor or spleen (bot- tom) volume dissected from mice vaccinated with ME49Δompdc/gra4, ME49Δompdc, or PBS, followed by implanted B16-F10 tumor cells. G) Represen- tative flow plots (left) and histogram (right) of CD4+ and CD8+ T cells within splenocytes from mice vaccinated with ME49Δompdc, ME49Δompdc/gra4, or PBS, followed by implanted B16-F10 tumor cells. H) Representative quantification of PD-1 expression in CD4+ (top) and CD8+ (bottom) T cells from mice vaccinated with ME49Δompdc, ME49Δompdc/gra4, or PBS, followed by implanted B16-F10 tumor cells. I) Representative plots (left) and histogram (right) of IFN-𝛾of CD4+ T and CD8+ T cells within splenocytes from mice vaccinated with ME49Δompdc, ME49Δompdc/gra4, or PBS, followed by implanted B16-F10 tumor cells. J) Macroscopic evaluation of B16-F10 tumors metastasis in the lungs of mice treated with PBS (isotype con-

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA): IFN-β in cell supernatants and mice serum was measured with the Mouse IFN-beta DuoSet ELISA kit (R&D SYSTEMS, Cat# DY8234-05) following the assay procedure.

Techniques: Gene Expression, Infection, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Oncogene

Article Title: Fibroblast growth factor receptor is a mechanistic link between visceral adiposity and cancer

doi: 10.1038/onc.2017.278

Figure Lengend Snippet: MFTF stimulates JB6 P + cell transformation. SKH-1 mice ( n =5/group) were fed either a HFD or LFD for 4 weeks. Visceral (parametrial and epididymal) adipose tissue was removed to make a filtered conditioned medium (MFTF). ( a ) Percentage of clones growing in soft agar (% colony formation) significantly increases in JB6 P + cells cultured with MFTF compared no treatment (control; Cont). No significant change in the percentage of colony formation in soft agar is observed in JB6 P − cells cultured with MFTF. ( b ) JB6 P + colonies growing in soft agar with HFD MFTF is significantly inhibited with proteinase K, but not with lipase, RNase A or DNase A. ( c ) JB6 P + colonies growing in soft agar decrease as MFTF is exposed to increasing temperatures for 30 min prior to treating the cells in agar. ( d ) Protein Profiler angiogenesis array of fat tissue filtrates of LFD-fed mice (top panel) and HFD-fed mice (bottom panel). HFD selectively upregulated protein levels of several key adipokines, hormones and growth factors in MFTF, versus those seen in MFTF of LFD-fed mice (boxed proteins). Dot intensity was analyzed by ‘Image J’ software. Data are labeled as the percent of the control (reference) dots located in the upper left hand corner of the arrays. Data are presented as mean±s.d. of values from triplicate. Statistical significance was determined using a one-way ANOVA (** P <0.01, *** P <0.001).

Article Snippet: Measurement of mouse adipokines and growth factors from fat tissue filtrates was performed using the Mouse Angiogenesis Antibody Array kit (R&D Systems Cat# ARY015).

Techniques: Transformation Assay, Clone Assay, Cell Culture, Control, Software, Labeling

Journal: Oncogene

Article Title: Fibroblast growth factor receptor is a mechanistic link between visceral adiposity and cancer

doi: 10.1038/onc.2017.278

Figure Lengend Snippet: Lipectomy reduces transformation-stimulating serum growth factors in HFD-fed mice. ( a ) SKH-1 mice were fed either a HFD or LFD for 2 weeks, and half the mice had their parametrial adipose tissue removed or received a sham operation ( n =20/group). After 33 weeks of UVB exposure, serum was isolated. Protein Profiler angiogenesis array of pooled sera of HFD-fed sham-operated mice (top panel) and HFD-fed lipectomized mice (bottom panel). HFD-fed mice that had the surgical removal of parametrial adipose tissue showed a decrease in several pro-inflammatory proteins in the circulation. Boxed proteins were those found to be both reduced with lipectomy and induced in the MFTF with HFD . Dot intensity was analyzed by ‘ImageJ’ software. Data are labeled as the percent of the control (reference) dots located in the upper left hand corner of the arrays. ( b ) Proteins found in a were tested for their transforming activity in the soft agar assay. HGF (10 ng/ml) and FGF2 (10 ng/ml) significantly stimulated colony formation in soft agar. 12- O -tetradecanoylphorbol-13-acetate (TPA, 10 ng/ml) was used as a positive control. ( c ) HGF and FGF2 levels in MFTF from were quantified by ELISA. HFD feeding increases the levels of both HGF and FGF2. ( d ) SKH-1 mice were treated as described in a . Serum was isolated and analyzed for HGF and FGF2 by ELISA. ( e ) Dose response of HGF and FGF2 on JB6 P + cell transformation. Data are presented as mean±s.d. of values from triplicates and statistical significance was determined using a one-way ANOVA ( b – e ) followed by a Tukey’s test for multiple comparisons ( d ) (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Measurement of mouse adipokines and growth factors from fat tissue filtrates was performed using the Mouse Angiogenesis Antibody Array kit (R&D Systems Cat# ARY015).

Techniques: Transformation Assay, Isolation, Software, Labeling, Control, Activity Assay, Soft Agar Assay, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Cell reports. Medicine

Article Title: CV1-secreting sCAR-T cells potentiate the abscopal effect of microwave ablation in heterogeneous tumors.

doi: 10.1016/j.xcrm.2025.101965

Figure Lengend Snippet: Figure 5. Released chemokines post-MWA by residual tumor are critical for the recruitment of macrophages that elicited the abscopal effect (A) Cytokine array from water-bathed (48C, 10 min) and control SK-HEP-1-GPC3 human liver cancer cells on days 1, 3, and 6 by bulk RNA sequencing, n = 3, one experiment. (B) CSF1, CCL26, and CXCL8 secretion in water-bathed supernatant of SK-HEP-1-GPC3 after 48 h by ELISA, n = 3 independent experiments. (C) Normalized migration ability of mouse macrophages cultured with conditioned supernatant of water-bathed or control SK-HEP-1-GPC3 cells, n = 3 inde- pendent experiments. (D–G) Relative Csf1 mRNA level by qPCR in control and Csf1 knockout SK-HEP-1-GPC3 cells, n = 3 independent experiments (D). Distant tumor (SK-HEP-1) growth followed by local MWA and local SK-HEP-1-GPC3 Csf1 knockout ± MAsCART in NOG mice, n = 5, one experiment (E and F). Distant tumor-infiltrating macrophages in different groups following local MWA and local SK-HEP-1-GPC3 Csf1 knockout ± MAsCART in NOG mice on day 6, n = 5, one experiment (G). Error bars are means ± SD (B, C, D, F, and G). *p < 0.05, **p < 0.01, ***p < 0.001 by t test for (C) and (D), two-way (F), or one-way (G) ANOVA with Tukey’s multiple comparisons test. See also Figure S4.

Article Snippet: N/A Chemicals, peptides, and recombinant proteins Penicillin-Streptomycin Sigma-Aldrich Cat#P4458; CAS: 8025-06-7 Puromycin Sigma-Aldrich Cat#P9620; CAS: 58-58-2 Lymphoprep Stem Cell Cat#07861; CAS: 66720-17-0 Human IL-2 Recombinant Protein PeproTech Cat#200-02-1MG Mouse IL-2 Recombinant Protein PeproTech Cat#212-12-1MG Mouse M-CSF Recombinant Protein PeproTech Cat#315-02-1MG Phorbol 12-myristate 13-acetate (PMA) MCE Cat#HY-18739 Mouse recombinant IL-4 PeproTech Cat#214-14 anti-human CD3/CD28 microbeads Thermo Fisher Cat#11131D anti-mouse CD3/CD28 microbeads Thermo Fisher Cat#11453D Protease Inhibitor Cocktail Thermo Fisher Cat#5892970001 D-luciferin Cayman Chemical Cat#2591-17-5; CAS: 2591-17-5 TRIzolTM Reagent Thermo Fisher Cat#15596018 Retronectin Takara Cat#T100B Critical commercial assays EasySepTM Release Human CD3 Positive Selection Kit Stem Cell Cat#17751 EasySepTM Mouse T cell Isolation Kit Stem Cell Cat#19851 EasySepTM Mouse F4/80 Positive Selection Kit Stem Cell Cat#100-0659 EasySepTM Release Human CD45 Positive Selection Kit for Humanized Mice Stem Cell Cat#100-0107 Human CSF-1 Quantikine ELISA Kit R&D Systems Cat#SMC00B Human CCL26 Quantikine ELISA Kit R&D Systems Cat#DCC260B (Continued on next page) Cell Reports Medicine 6, 101965, February 18, 2025 e1

Techniques: Control, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Migration, Cell Culture, Knock-Out

Journal: Cell reports

Article Title: Tim-4 reprograms cholesterol metabolism to suppress antiviral innate immunity by disturbing the Insig1-SCAP interaction in macrophages.

doi: 10.1016/j.celrep.2022.111738

Figure Lengend Snippet: Figure 1. Tim-4 represses the antiviral response in macrophages (A–H) PEMs were isolated from WT or Tim-4 KO mice and infected with VSV (0.1 multiplicity of infection [MOI]) for the indicated times. (A) qRT-PCR and ELISA were performed to analyze Ifnb mRNA (left) and IFN-b secretion (right), respectively. (B) Mxa and Cxcl10 expression were detected by qRT-PCR. (C) Immunoblotting was used to detect IRF3 and IRF3 phosphorylation. The relative ratios of p-IRF3 to total IRF3 are indicated below. (D) VSV mRNA levels were measured by qRT-PCR. (E) VSV replication was assayed by observing VSV-GFP under fluorescence microscopy in WT and Tim-4 KO PEMs infected with VSV for 12 h (fluorescence, upper; bright field, bottom). Scale bars, 100 mm. VSV-GFP fluorescence intensity was quantified over 10 images using ImageJ. Fluorescence intensity was measured for the entire field of view. The results are shown as mean ± SEM (n = 10) (right). (F) Flow cytometry analysis of VSV-GFP fluorescence intensity. (G) Plaque assay of VSV titers. PFU, plaque-forming units. (H) Immunoblotting analysis of VSV-G protein levels. The relative ratios of VSV-G and GAPDH are indicated below. RAW264.7 cells were transfected with empty or Tim-4 overexpression plasmid for 48 h, followed by infection with VSV (1 MOI) for another 8 (I–K) or 12 h (L–O). (I) The expression of IFN-b was detected by qRT-PCR (left) and ELISA (right). (J) qRT-PCR analysis of Mxa and Cxcl10 mRNA expression. (K) Immunoblotting analysis of IRF3 and phosphorylated IRF3. The relative ratios of p-IRF3 to total IRF3 are indicated below.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER MinuteTM Golgi Apparatus Enrichment Kit Invent biotech Cat# GO-037 Mouse IFN-beta DuoSet ELISA kit R&D Systems Cat# DY8234-05 IFN-b ELISA kit Valukine Cat# VAL612 KOD -Plus- Mutagenesis Kit TOYOBO Cat# SMK-101 BCA assay Beyotime Biotechnology Cat# P0009 AmplexTM Red Cholesterol Assay Kit Thermo Fisher Cat# A12216 Deposited data RNA-seq data This paper GEO: GSE214346 Experimental models: Cell lines HEK293T Cell Bank of the Chinese Academy of Sciences GNHu17 HEK293 Cell Bank of the Chinese Academy of Sciences GNHu43 HeLa Cell Bank of the Chinese Academy of Sciences TCHu187 RAW264.7 ATCC TIB-71 THP-1 Cell Bank of the Chinese Academy of Sciences SCSP-567 Experimental models: Organisms/strains Mouse: Tim-4 KO RIKEN BioResource Research Center BRC No. RBRC04895 Mouse: Wild type Vital River Laboratory Animal Technology N/A Oligonucleotides For primers sequence for qPCR see Table S1 This paper N/A si-Srebf2:50-GGCCAUUGAUUACAUCAAATT-30 GenePharma N/A siRNA universal negative control GenePharma N/A Recombinant DNA pcDNA3-HA-Tim-4 Liu et al.35 N/A pcDNA3-HA-Tim-4 PS mut Liu et al.35 N/A pcDNA3-HA-Tim-4 DIgV Liu et al.35 N/A pcDNA3-HA-Tim-4 Dmucin Liu et al.35 N/A pcDNA3-HA-Tim-4 Dcyto Liu et al.35 N/A pCDNA3.4-SCAP-33Flag Miaoling bio Cat# P20386 pCDNA3.4-SCAP Y298C This paper N/A pcDNA3.1-Insig1-myc Research Cloud Biology N/A p3-Flag-NSREBP2 Gift from Prof. Xuetian Yue (Shandong University) N/A plenti-33Flag SREBP2 Gift from Prof. Jian Li (Shandong University) N/A LDLR reporter Gift from Prof. Jian Li (Shandong University) N/A HMGCR reporter Gift from Prof. Jian Li (Shandong University) N/A pmcherry-Sec61b-C1 This paper N/A Software and algorithms GraphPad Prism version 8.0 GraphPad Software Inc www.graphpad.com ZEN 2.3 lite Zeiss www.zeiss.com (Continued on next page) e3 Cell Reports 41, 111738, November 29, 2022

Techniques: Isolation, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Phospho-proteomics, Microscopy, Fluorescence, Flow Cytometry, Plaque Assay, Transfection, Over Expression, Plasmid Preparation

Journal: Cell reports

Article Title: Tim-4 reprograms cholesterol metabolism to suppress antiviral innate immunity by disturbing the Insig1-SCAP interaction in macrophages.

doi: 10.1016/j.celrep.2022.111738

Figure Lengend Snippet: Figure 2. Tim-4 deficiency in macrophages protects the host from viral infection in vivo WT and Tim-4 KO mice were infected with VSV by intraperitoneal injection (5 3 107 PFU per mouse) for 48 h (PBS group, n = 3; VSV group, n = 4). (A) qRT-PCR analysis of Ifnb mRNA expression in the lung, liver, and spleen. (B and C) ELISA of IFN-b in lung and liver tissue homogenates (B) and serum (C). (D) qRT-PCR was used to determine the relative VSV mRNA levels in lung and liver tissues. (E) Plaque assay of VSV titers in lung and liver homogenates. (F) VSV-G protein levels in lung and liver tissues were examined by immunoblotting analysis. The relative ratios of VSV-G to GAPDH are marked below.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER MinuteTM Golgi Apparatus Enrichment Kit Invent biotech Cat# GO-037 Mouse IFN-beta DuoSet ELISA kit R&D Systems Cat# DY8234-05 IFN-b ELISA kit Valukine Cat# VAL612 KOD -Plus- Mutagenesis Kit TOYOBO Cat# SMK-101 BCA assay Beyotime Biotechnology Cat# P0009 AmplexTM Red Cholesterol Assay Kit Thermo Fisher Cat# A12216 Deposited data RNA-seq data This paper GEO: GSE214346 Experimental models: Cell lines HEK293T Cell Bank of the Chinese Academy of Sciences GNHu17 HEK293 Cell Bank of the Chinese Academy of Sciences GNHu43 HeLa Cell Bank of the Chinese Academy of Sciences TCHu187 RAW264.7 ATCC TIB-71 THP-1 Cell Bank of the Chinese Academy of Sciences SCSP-567 Experimental models: Organisms/strains Mouse: Tim-4 KO RIKEN BioResource Research Center BRC No. RBRC04895 Mouse: Wild type Vital River Laboratory Animal Technology N/A Oligonucleotides For primers sequence for qPCR see Table S1 This paper N/A si-Srebf2:50-GGCCAUUGAUUACAUCAAATT-30 GenePharma N/A siRNA universal negative control GenePharma N/A Recombinant DNA pcDNA3-HA-Tim-4 Liu et al.35 N/A pcDNA3-HA-Tim-4 PS mut Liu et al.35 N/A pcDNA3-HA-Tim-4 DIgV Liu et al.35 N/A pcDNA3-HA-Tim-4 Dmucin Liu et al.35 N/A pcDNA3-HA-Tim-4 Dcyto Liu et al.35 N/A pCDNA3.4-SCAP-33Flag Miaoling bio Cat# P20386 pCDNA3.4-SCAP Y298C This paper N/A pcDNA3.1-Insig1-myc Research Cloud Biology N/A p3-Flag-NSREBP2 Gift from Prof. Xuetian Yue (Shandong University) N/A plenti-33Flag SREBP2 Gift from Prof. Jian Li (Shandong University) N/A LDLR reporter Gift from Prof. Jian Li (Shandong University) N/A HMGCR reporter Gift from Prof. Jian Li (Shandong University) N/A pmcherry-Sec61b-C1 This paper N/A Software and algorithms GraphPad Prism version 8.0 GraphPad Software Inc www.graphpad.com ZEN 2.3 lite Zeiss www.zeiss.com (Continued on next page) e3 Cell Reports 41, 111738, November 29, 2022

Techniques: Infection, In Vivo, Injection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Plaque Assay, Western Blot