Journal: Cell Death & Disease

Article Title: Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling

doi: 10.1038/s41419-022-04771-3

Figure Lengend Snippet: A Cytokine antibody microarray analysis of brainstem of ICR mice infected with MP4 virus. The ICR mice at 7-day of age were orally infected with or without a 50% lethal dose of a mouse-adapted EV71 strain (MP4) virus and the brainstem tissues were harvested for the analysis at 7 days post-infection (dpi). B qRT-PCR analysis of IL-12p40, IL12A, IL23, and IFNγ mRNA levels at 7 dpi in the brainstem of ICR mice infected with or without MP4 virus. Values are represented as mean ( n = 4). Significantly different from control group: ** P < 0.01 by unpaired t test. C Western blot analysis of IL-12p40 protein levels in the brainstem of hSCARB2-Tg mice infected with EV71 (3 × 10 4 PFU/mouse). The hSCARB2-Tg (TG) and non-Tg (WT) mice at 7-day of age were injected s.c. with 3 × 10 4 pfu of 5746 (C2) strain of EV71. The brainstem tissues ( n = 3) were harvested at the indicated dpi for western blot analysis. D, Temporal expression of IL-12p40 in the brainstem of hSCARB2-Tg (TG) and non-TG (WT) mice infected with EV71. Time-course qRT-PCR analysis of IL-12p40 at the indicated dpi. Values are represented as mean ( n = 4). Significantly different from control group: * P < 0.05, **P < 0.01 by unpaired t test. E Representative photomicrographs of anti-GFAP/IL-12p40 immunostaining in the brainstem regions. Scale bars, 50 μm. Time-course qRT-PCR analysis of IL-12p40 mRNA levels in glial and neuronal cells. U87-MG or SH-SY5Y cells were infected with or without EV71 (MOI = 1) and harvested at the indicted hpi for the analysis ( F ). Primary microglia and neurons isolated from hSCARB2-Tg mice were infected with or without EV71 (MOI = 3) and assayed at the indicted hours post-infection ( G ). Values are represented as mean ( n = 4–6). *** P < 0.001 by One-Way ANOVA.

Article Snippet: An IL-12p40 ELISA assay (Elabscience Biotechnology) was used to detect serum IL-12p40 levels in the EV71-infected hSCARB2-Tg mice.

Techniques: Microarray, Infection, Virus, Quantitative RT-PCR, Control, Western Blot, Injection, Expressing, Immunostaining, Isolation

Journal: Cell Death & Disease

Article Title: Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling

doi: 10.1038/s41419-022-04771-3

Figure Lengend Snippet: A Kaplan-Meier survival curve of EV71-infected hSCARB2-Tg mice receiving treatment with anti-IL-12p40 antibody or control IgG. The 7-day-old hSCARB2-Tg mice were injected s.c. with 3 × 10 4 pfu of EV71 and subsequently intraperitoneally injected with anti-IL12p40 antibody (2 ug/mouse) or IgG (Ctrl) at 2 and 4 dpi. Significantly different from control group at p < 0.01 by Log Rank test. B Representative photomicrographs and quantification of anti-GFAP immunostaining in the brainstem regions of the EV71-infected hSCARB2-Tg mice treated with anti-IL-12p40 antibody or control IgG. Scale bars, 50 μm. The GFAP-positive signals in five consecutive sections per animal was quantified by ImageJ and presented as mean ( n = 5, right panel). Significantly different from control group: *** P < 0.01 by unpaired t test. C qRT-PCR analysis of TNFa and IL6 mRNA levels in the brainstem of the EV71-infected hSCARB2-Tg mice treated with anti-IL-12p40 antibody or control IgG. Values are represented as mean ( n = 4). Significantly different from control group at * p < 0.05 by unpaired t test. D Progressive limb paralysis in the EV71-infected hSCARB2-Tg mice treated with anti-IL-12p40 antibody or control IgG. Limb paralysis (LP) is defined as the rigidness of mouse legs which are hesitate to move. For scoring LP; 5 = severe front and rear LP and no movement, 4 = moderate two rear LP and hesitant movement, 3 = one rear LP with bending legs, 2 = mild rear limb bended, 1 = slightly rear limb bended, 0 = normal movement. Significantly different from control group: p < 0.001 by unpaired t test. E The configuration of temporal sequential order of the occurrence of IL-12p40 expression, limb paralysis, and mortality in the EV71-infected hSCARB2-Tg mice.

Article Snippet: An IL-12p40 ELISA assay (Elabscience Biotechnology) was used to detect serum IL-12p40 levels in the EV71-infected hSCARB2-Tg mice.

Techniques: Infection, Control, Injection, Immunostaining, Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling

doi: 10.1038/s41419-022-04771-3

Figure Lengend Snippet: A Temporal expression pattern of TLR9 in the brainstem of hSCARB2-Tg (TG) and non-TG (WT) mice infected with EV71. qRT-PCR analysis of TLR9 at the indicated dpi. Values are represented as mean ( n = 3). Significantly different from control group: * P < 0.05, ** P < 0.01 by unpaired t test. B Representative photomicrographs of TLR9 (green) colocalized with IL-12p40 (red) in the brainstem regions of mice infected with EV71 at 4dpi. Scale bars, 40 μm. C qRT-PCR analysis of IL-12p40 expression levels in the brainstem of the EV71-infected hSCARB2-Tg mice with the treatment ODN2088 or control IgG. Values are represented as mean ( n = 5). Significantly different from control group: ** P < 0.01 by unpaired t test. D EV71-induced limb paralysis at indicated dpi in hSCARB2-Tg mice treated with or without ODN2088. Significantly different from control group: p < 0.001 by unpaired t test. E Representative photomicrographs of anti-GFAP immunostaining in the brainstem regions of the EV71-infected hSCARB2-Tg mice treated with or without ODN2088 at 3 dpi. Scale bars, 50 μm. The GFAP-positive signals in five consecutive sections per animal ( n = 5) was quantified by ImageJ and presented as mean ± SD (right panel). Significantly different from control group: *** P < 0.001 by two-tailed unpaired t test. F qRT-PCR analysis of TNFα and IL-6 mRNA levels in the brainstem of the EV71-infected hSCARB2-Tg mice treated with or without ODN2088. Values are represented as mean ( n = 5). Significantly different from control group: * p < 0.05 by two-tailed unpaired t test. G Histopathological examination of brainstem in the EV71-infected hSCARB2-Tg mice treated with ODN2088 or ctrl. Asterisks (∗) on the field of pathological foci with monocytes/macrophages infiltration. Number of lesion in five consecutive sections per animal ( n = 5) was quantified and presented as the mean. Significantly different from control group, ** P < 0.01 by unpaired t test.

Article Snippet: An IL-12p40 ELISA assay (Elabscience Biotechnology) was used to detect serum IL-12p40 levels in the EV71-infected hSCARB2-Tg mice.

Techniques: Expressing, Infection, Quantitative RT-PCR, Control, Immunostaining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling

doi: 10.1038/s41419-022-04771-3

Figure Lengend Snippet: A Graphical illustration of how different subunits of IL-12 interact with specific cell surface receptors in controlling distinct downstream transcription factors and signaling events. B qRT-PCR analysis of iNOS expression levels in the brainstem of hSCARB2-Tg (TG) or non-TG (WT) mice during the time course of EV71 infection. Values are represented as mean ( n = 3). Significantly different from control group: ** P < 0.01 by unpaired t test. C qRT-PCR analysis of iNOS expression levels in the EV71-infected hSCARB2-Tg mice treated with or without anti-IL-12p40 antibody ( n = 5). D qRT-PCR analysis of iNOS expression levels in the EV71-infected U87-MG cells treated with or without anti-IL-12p40 antibody ( n = 3). Effect of NO level on EV71-induced cell viability. SH-SY5Y and primary neuronal cells were pre-treated with or without NO donor SNAP ( E , F ) or Nitric oxide synthase inhibitor LNNA ( G , H ) 1 h before infection with EV71 (MOI = 1 for SH-SY5Y and MOI = 3 for primary neuron). WST-1 analysis was conducted at 9 hpi. Values are represented as mean ( n = 4). Significantly different from control group at * p < 0.05, ** p < 0.01 by unpaired t test.

Article Snippet: An IL-12p40 ELISA assay (Elabscience Biotechnology) was used to detect serum IL-12p40 levels in the EV71-infected hSCARB2-Tg mice.

Techniques: Quantitative RT-PCR, Expressing, Infection, Control

Journal: Cell Death & Disease

Article Title: Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling

doi: 10.1038/s41419-022-04771-3

Figure Lengend Snippet: Severe EV71 infection induces an acute IL-12p40 response through intracellular sensor TLR9 in glia cells of the brainstem, subsequently generating neurotoxic NO causing encephalitis. These findings provide support for the therapeutic value of blocking the TLR9 mediated IL-12p40 response in the treatment of severe EV71 infection-induced brainstem encephalitis.

Article Snippet: An IL-12p40 ELISA assay (Elabscience Biotechnology) was used to detect serum IL-12p40 levels in the EV71-infected hSCARB2-Tg mice.

Techniques: Infection, Blocking Assay

Journal: Cell Reports Medicine

Article Title: LTA4H improves the tumor microenvironment and prevents HCC progression via targeting the HNRNPA1/LTBP1/TGF-β axis

doi: 10.1016/j.xcrm.2025.102000

Figure Lengend Snippet: Downregulation of LTA4H was associated with poor prognosis in patients with HCC (A) IHC detection of LTA4H in human HCC tissues. (B and C) TMA analysis of LTA4H in HCC and para-tumor tissues. (D and E) Western blot (WB) and quantification of LTA4H in clinical HCC samples. (F) qPCR detection of LTA4H in clinical HCC samples. (G–J) WB, qPCR, and IHC detection of LTA4H in the DEN-induced mouse HCC model. (K) ELISA detection of LTB4 in DEN-induced mouse HCC models. (L) ELISA detection of LTB4 in clinical HCC samples. (M) Pearson correlation of LTB4 with LTA4H mRNA and protein levels in clinical HCC samples. (N) Representative images of tumors with low and high LTA4H expression in the TMA HCC cohort. (O and P) Overall survival (OS) and recurrence-free survival (RFS) according to LTA4H level of patients with HCC in the TMA HCC cohort ( n = 133). (Q and R) Comparison of LTA4H mRNA and protein levels in tumoral and adjacent tissues in the CHCC-HBV cohort. (S) Pearson correlation of LTA4H mRNA and protein levels in the CHCC-HBV cohort. (T) OS analysis according to the LTA4H protein level in the CHCC-HBV cohort. (A, J, and N) Scale bar: 100 μm. (B) Scale bar: 300 μm. (C) p value was calculated by Wilcoxon rank-sum test ( n = 133), data represent mean ± SD. (E, F, and L) p value was calculated by paired Wilcoxon signed-rank test ( n = 10). (O, P, and T) p value was calculated by log rank test. (H, I, and K) p value was calculated by Student’s t test ( n = 3), data represent mean ± SD. (Q and R) p value was calculated by Wilcoxon rank-sum test ( n = 159), the line and box represent median and upper and lower quartiles, respectively. All the replicates represent biological replicates. See also Tables S1 and .

Article Snippet: The levels of total TGF-β and active TGF-β were determined using a mouse TGF-β ELISA kit (R&D Systems, DB100C) according to the manufacturer’s instructions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Comparison

Journal: Cell Reports Medicine

Article Title: LTA4H improves the tumor microenvironment and prevents HCC progression via targeting the HNRNPA1/LTBP1/TGF-β axis

doi: 10.1016/j.xcrm.2025.102000

Figure Lengend Snippet: Hepatocyte-specific LTA4H deficiency promotes M2-like macrophage polarization by upregulating LTBP1 expression and TGF-β secretion (A) Volcano plot of differentially expressed and secreted genes in LTA4H KO vs. LTA4H f/f hepatocytes ( Figure 3 A). (B) Transcript level of Ltbp1 in LTA4H f/f and LTA4H KO hepatocytes ( n = 3). (C and D) mRNA and protein levels of LTBP1 in liver tissues of LTA4H f/f , LTA4H Δhep , and LTA4H KO mice ( n = 5). (E–G) mRNA and protein levels of LTBP1 in Hepa1-6 cells with LTA4H overexpression or knockout ( n = 3). (H and I) ELISA for secreted TGF-β in supernatants of Hepa1-6 cells with LTA4H overexpression or knockout ( n = 3). (J and K) ELISA for active and total TGF-β serum levels in LTA4H f/f , LTA4H Δhep , and LTA4H KO DEN-induced HCC models ( n = 4). (L) The coculture model of BMDMs and CM from scramble or LTA4H KO Hepa1-6 cells, CM pretreated with HCl/NaOH. (M and N) Transcript levels of Arg1 and Mrc1 in BMDMs under different treatments ( n = 3). (O) CD206 immunofluorescence in BMDMs from coculture system under various treatments. Scale bar: 200 μm. (P) Quantification of CD206 + macrophage in BMDMs from coculture system under various treatments ( n = 3). (Q) mIHC staining for LTA4H, LTBP1, CD206, and CD68 in HCC tissues with low or high LTA4H expression from the TMA HCC cohort. Scale bar: 200 μm, enlarged images scale bar: 20 μm. (R) Quantification and Pearson correlation among LTA4H, LTBP1, and CD206 + macrophages in the TMA HCC cohort. (S) Liver images and H&E staining of orthotopic HCC tumors (Hepa1-6 scramble and LTA4H KO) with or without macrophage depletion. Scale bar: 4 mm. (T) Tumor burden comparison across groups, determined by tumor-to-liver area ratio in sections ( n = 5). (B, E, and H) p value was calculated by Student’s t test, data represent mean ± SD. (C, F, I–K, M, N, P, and T) p values were calculated by one-way ANOVA with Tukey’s multiple comparison analysis, data represent mean ± SD. All the replicates represent biological replicates. See also Figures S5 and .

Article Snippet: The levels of total TGF-β and active TGF-β were determined using a mouse TGF-β ELISA kit (R&D Systems, DB100C) according to the manufacturer’s instructions.

Techniques: Expressing, Over Expression, Knock-Out, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Comparison

Journal: Cell Reports Medicine

Article Title: LTA4H improves the tumor microenvironment and prevents HCC progression via targeting the HNRNPA1/LTBP1/TGF-β axis

doi: 10.1016/j.xcrm.2025.102000

Figure Lengend Snippet:

Article Snippet: The levels of total TGF-β and active TGF-β were determined using a mouse TGF-β ELISA kit (R&D Systems, DB100C) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Microarray, Recombinant, Lysis, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Reverse Transcription, Activity Assay, Immunoprecipitation, Extraction, Purification, In Vitro, Transfection, Sequencing, Mass Cytometry, ChIP-sequencing, Transgenic Assay, Software

Journal: Infection and Immunity

Article Title: cDNA Microarray Analysis of Host-Pathogen Interactions in a Porcine In Vitro Model for Toxoplasma gondii Infection

doi: 10.1128/iai.00386-05

Figure Lengend Snippet: FIG. 1. Transcriptional profile of porcine kidney epithelial (PK13) cells infected with T. gondii for 0 to 72 h. (A) Hierarchical clustering of the expression pattern of cDNAs representing 401 known genes, analyzed by microarray. Each row represents an individual cDNA element spotted on the array, and each column represents the expression states of cDNAs at a particular time point postinfection. Each expression data point represents the ratio of the fluorescence intensity of the cDNA from T. gondii-infected cells to the fluorescence intensity of the cDNA from noninfected control cells and is the average value of 12 data points for each time point of the experiment. The cluster is subdivided into two major groups, indicated by roman numerals at the left, consisting of genes that were repressed (i) (green) and genes that were induced (ii) (red). Genes whose expression did not change are colored black on the gene tree. (B) Enlarged view of the cluster from panel A, showing the genes that are modulated at 0 to 72 h postinfection.

Article Snippet: Porcine kidney epithelial cell line PK13 (CRL6489; ATTC, Manassas, VA) was cultured in 75-cm2 Greiner tissue culture flasks containing 15 ml of Dulbecco’s modified Eagle’s medium (DMEM) with 4.0 mM L-glutamine, 4.5g/liter glucose, and 1.0 mM sodium pyruvate (ATCC 30-2002) and supplemented with 10% heat-inactivated fetal bovine serum (HIFBS) (ATCC 30-2020).

Techniques: Infection, Expressing, Microarray, Control

Journal: Infection and Immunity

Article Title: cDNA Microarray Analysis of Host-Pathogen Interactions in a Porcine In Vitro Model for Toxoplasma gondii Infection

doi: 10.1128/iai.00386-05

Figure Lengend Snippet: FIG. 2. Effect of T. gondii infection on c-Fos activity (A) and NF- B2 activity (B). Activation assays were performed as described in the text, using whole-cell lysates isolated from T. gondii-infected PK13 cells and uninfected PK13 control cells at 1 h, 2 h, 4 h, 6 h, 24 h, 48 h, and 72 h postinfection. Transcription factor activation in infected cells is reported relative to that observed in uninfected control cells. Error bars indicate standard deviations.

Article Snippet: Porcine kidney epithelial cell line PK13 (CRL6489; ATTC, Manassas, VA) was cultured in 75-cm2 Greiner tissue culture flasks containing 15 ml of Dulbecco’s modified Eagle’s medium (DMEM) with 4.0 mM L-glutamine, 4.5g/liter glucose, and 1.0 mM sodium pyruvate (ATCC 30-2002) and supplemented with 10% heat-inactivated fetal bovine serum (HIFBS) (ATCC 30-2020).

Techniques: Infection, Activity Assay, Activation Assay, Isolation, Control

Journal: PLoS Pathogens

Article Title: Microglia Are Mediators of Borrelia burgdorferi –Induced Apoptosis in SH-SY5Y Neuronal Cells

doi: 10.1371/journal.ppat.1000659

Figure Lengend Snippet: (A) Five of the ten canonical pathways most affected during co-culture of microglia with B. burgdorferi involved inflammatory signaling (in bold) and occurred in cells isolated from each of two rhesus macaques sampled. The number in parenthesis indicates the pathway order of significance in the 2nd animal. Bar graph represents the ratio of gene expression in microglia co-cultured with B. burgdorferi as compared to control microglia cultured in medium alone. Line graph represents significance as –log (p-value) where p<0.05. (B) Microarray analysis of the triggering receptor expressed in myeloid cells (TREM1) pathway indicates, through transcript up-regulation of numerous pro-inflammatory genes including MCP-1 (CCL2), IL-6 and lL-8, the tendency for a sustained microglia-generated inflammatory response to B. burgdorferi. Values were obtained using Ingenuity Pathways Analysis (IPA) software, version 6. Minimum fold change was ≥2.0 with significance of p<0.05. All genes in this pathway were represented on the array chips. Red: increased gene expression. Green: decreased gene expression. The results displayed in sections (A) and (B) represent findings from animal number 1. Individual transcript changes for animals 1 and 2 can be accessed through the online supplemental data ( and ).

Article Snippet: We obtained CCL3 and CCL4 ELISA DuoSet kits and the MCP-1 (CCL2) kit from R&D Systems.

Techniques: Co-Culture Assay, Isolation, Gene Expression, Cell Culture, Control, Microarray, Generated, Software

Journal: PLoS Pathogens

Article Title: Microglia Are Mediators of Borrelia burgdorferi –Induced Apoptosis in SH-SY5Y Neuronal Cells

doi: 10.1371/journal.ppat.1000659

Figure Lengend Snippet: Gene transcripts significantly affected by Borrelia burgdorferi (Bb) in microglia.

Article Snippet: We obtained CCL3 and CCL4 ELISA DuoSet kits and the MCP-1 (CCL2) kit from R&D Systems.

Techniques: Derivative Assay

Journal: PLoS Pathogens

Article Title: Microglia Are Mediators of Borrelia burgdorferi –Induced Apoptosis in SH-SY5Y Neuronal Cells

doi: 10.1371/journal.ppat.1000659

Figure Lengend Snippet: Molecules associated with TREM1 signaling in microglia co-cultured in the presence of B. burgdorferi for 5 days.

Article Snippet: We obtained CCL3 and CCL4 ELISA DuoSet kits and the MCP-1 (CCL2) kit from R&D Systems.

Techniques:

Journal: PLoS Pathogens

Article Title: Microglia Are Mediators of Borrelia burgdorferi –Induced Apoptosis in SH-SY5Y Neuronal Cells

doi: 10.1371/journal.ppat.1000659

Figure Lengend Snippet: Confirmation of rhesus array results using QRT-PCR.

Article Snippet: We obtained CCL3 and CCL4 ELISA DuoSet kits and the MCP-1 (CCL2) kit from R&D Systems.

Techniques: