Journal: EMBO Molecular Medicine

Article Title: Immune training enhances anti-viral responses and improves outcomes in Pax5 −/+ mice susceptible to chronic infection

doi: 10.1038/s44321-025-00208-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Ly6C , Novus Biologicals , NBP1-28046.

Techniques: Recombinant, Staining, Red Blood Cell Lysis, Sequencing, Conjugation Assay, Software, Cell Culture, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1 *

doi: 10.1074/jbc.M113.546077

Figure Lengend Snippet: Hook1 was a new binding protein of SHP2. A , co-immunoprecipitation assay to verify the interaction of overexpressed full-length SHP2 (MYC-SHP2) or PTP domain of SHP2 (MYC-SHP2-PTP) with FLAG-Hook1 in HEK293T cells. B , co-immunoprecipitation assay to verify the endogenous interaction of SHP2 with Hook1 in HEK293T cells.

Article Snippet: The Hook1 (NM_015888) human cDNA clone (cat. no. SC114574) was from OriGene (Rockville, MD).

Techniques: Binding Assay, Co-Immunoprecipitation Assay

Journal: The Journal of Biological Chemistry

Article Title: SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1 *

doi: 10.1074/jbc.M113.546077

Figure Lengend Snippet: Hook1 negatively regulated EMT. A , quantification of the mRNA levels of CDH1 , Hook1, Hook2 , and Hook3 in A549 cells with TGFβ1 treatment (5 ng/ml) for 0, 3, 6, and 12 h (**, p < 0.01). B , Western blot analysis of the expression levels of Hook1 after treatment with TGFβ1 (5 ng/ml) for 48 h. C , Hook1 was knockdown after Hook1siRNA transfection in A549 cells. D , cell adhesion in control siRNA and Hook1siRNA-treated A549 cells. E , Western blot analysis of the effects of Hook1 overexpression on EMT markers (E-Cad and VIM) and ERK, Smad2, Smad3 activation in A549 cells with or without TGFβ1 treatment (5 ng/ml) for 24 h. F , QPCR analysis of EMT markers ( CDH1 , CDH2 , VIM , MMP9 , and COL1A1 ) under the same conditions as E . ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.01 between vector and Hook1. G , transwell assays to assess the migration of control and Hook1-transfected A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 16 h. Scale bar, 100 μm. ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.01 between vector and Hook1. H , QPCR analysis of EMT-related markers ( CDH1 , FN1 , ZEB1 , and COL1A1 ) in control siRNA and Hook1siRNA-transfected A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 24 h. ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.05 between CtrlsiRNA and Hook1siRNA. I, transwell assays to assess the migration of control and Hook1siRNA-treated A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 16 h. Scale bar, 100 μm. ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.05 between CtrlsiRNA and Hook1siRNA.

Article Snippet: The Hook1 (NM_015888) human cDNA clone (cat. no. SC114574) was from OriGene (Rockville, MD).

Techniques: Western Blot, Expressing, Knockdown, Transfection, Control, Over Expression, Activation Assay, Plasmid Preparation, Migration

Journal: The Journal of Biological Chemistry

Article Title: SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1 *

doi: 10.1074/jbc.M113.546077

Figure Lengend Snippet: Interaction of SHP2-NSH2 and PTP domains with Hook1. A , co-immunoprecipitation assay to verify the direct interaction of FLAG-Hook1C with either MYC-SHP2-NSH2 or MYC-SHP2-PTP in HEK293T cells. B , PTP activity assay to test SHP2 activity in immunocomplex from control siRNA or Hook1siRNA-transfected HEK293T cells, with or without EGF (50 ng/ml) stimulation for 15 min. ***, p < 0.005 between Ctrl and EGF; &, p < 0.05 between CtrlsiRNA and Hook1siRNA. C, PTP activity assay to test SHP2 activity in immunocomplex from A549 cells with or without TGFβ1 (5 ng/ml) stimulation for 24 h (***, p < 0.005). D , QPCR analysis of mRNA levels of EMT markers ( Snail1 , COL1A1 , FN1 ) in vector, Hook1, SHP2 E76V -transfected, Hook1 and E76V co-transfected A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 24 h. ***, p < 0.005 between with and without TGFβ1 treatment; &, p < 0.05 between SHP2E76V only and SHP2E76V plus Hook1. E , schematic model of the interaction between SHP2 and Hook1 in EMT. Hook1 locks the auto-inhibition caused by the intramolecular interaction between N-SH2 and PTP. Upon TGFβ1 treatment, Hook1 is reduced, and this weakens the interaction between N-SH2 and PTP, leading to the full activation of SHP2 by other molecules, including substrates.

Article Snippet: The Hook1 (NM_015888) human cDNA clone (cat. no. SC114574) was from OriGene (Rockville, MD).

Techniques: Co-Immunoprecipitation Assay, Activity Assay, Control, Transfection, Plasmid Preparation, Inhibition, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via mTORC2/HK1-mediated metabolic rewiring

doi: 10.1016/j.jbc.2023.105518

Figure Lengend Snippet: Deficiency of mTORC2 suppressed the stronger trained immunity in alveolar macrophages after mucosal rBCG PPE27 vaccination. A , immunoblot analysis for p-AKT(S473), total AKT, HK1, and HK2 in cell lysates from wild type alveolar macrophages exposed to 10 ng/ml LPS, 100 μg/ml β-glucan, BCG, or rBCG PPE27 (M.O.I:1:10) at indicated time post-training ( n = 2). B , levels of p-AKT(S473), total AKT, HK2, and RICTOR in cell lysates from RICTOR Ctrl or RICTOR CKO alveolar macrophages on 1 day after trained with rBCG PPE27 (M.O.I:1:10) ( n = 2). ( C – F ) glucose consumption, lactate production ( C ), OCR and ECAR ( D ), H3K4me3 enrichment ( E ), cytokines production ( F ) of RICTOR Ctrl or RICTOR CKO alveolar macrophages on 6 days following the depicted scheme ( n = 3–6). G , in vivo training mouse model via mucosal BCG or rBCG PPE27 vaccination for metabolic and epigenetic changes analysis. ( H – J ) lactate produced ( H ), H3K4me3 enrichment of Tnfa ( I ), and TNF-α production ( J ) of BMDMs or alveolar macrophages after restimulation in vitro with LPS according to F ( n = 3–5). In ( C , E , F , H and J ), means ± SEM of data are shown. ∗ p < 0.05, ∗∗ p < 0.01 by Student’s t test.

Article Snippet: Rapamycin-insensitive companion of mTOR ( RICTOR) fl/fl mice, HK1 fl/fl mice, LysM - Cre -expressing mice, and C57 black 6 (C57BL/6) mice were obtained from Cyagen Bio Co, Ltd (Shanghai, China).

Techniques: Western Blot, In Vivo, Produced, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via mTORC2/HK1-mediated metabolic rewiring

doi: 10.1016/j.jbc.2023.105518

Figure Lengend Snippet: HK1 deficiency abolished the metabolic and epigenetic changes in alveolar macrophages after mucosal rBCG PPE27 vaccination. A , immunoblot analysis for p-AKT (S473) and HK1 in cell lysates from HK1 Ctrl or HK1 CKO alveolar macrophages 24 h post stimulated with BCG or rBCG PPE27 (M.O.I: 1:10) ( n = 2). ( B – D ) glucose-6-p, citrate and lactate ( B ), OCR and ECAR ( C ), and H3K4me3 enrichment ( D ) produced by HK1 Ctrl or HK1 CKO alveolar macrophages on 6 days after training ( n = 4–6). ( E ) cytokines production of HK1 Ctrl or HK1 CKO alveolar macrophages trained by BCG or rBCG PPE27 after restimulation in vitro with LPS ( n = 6). F , in vivo training mouse model via intratracheal BCG or rBCG PPE27 vaccination. G to I , lactate levels, H3K4me3 enrichment, and TNF-α production according to F ( n = 3–5). In ( B , D , E , G , H and I ), means ± SEM of data are shown. ∗ p < 0.05 by Two-tailed Student’s t test. n.s. p > 0.05.

Article Snippet: Rapamycin-insensitive companion of mTOR ( RICTOR) fl/fl mice, HK1 fl/fl mice, LysM - Cre -expressing mice, and C57 black 6 (C57BL/6) mice were obtained from Cyagen Bio Co, Ltd (Shanghai, China).

Techniques: Western Blot, Produced, In Vitro, In Vivo, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via mTORC2/HK1-mediated metabolic rewiring

doi: 10.1016/j.jbc.2023.105518

Figure Lengend Snippet: Mucosal rBCG PPE27 vaccination confers enhanced protection against Mycobacterium infection dependent on mTORC2/HK1-mediated trained immunity. A , timeline of animal vaccination, infection and killing. A total of 3 to 6 mice per group were immunized with BCG or rBCG PPE27 (2 × 10 5 CFUs) in 100 μl of PBS via the mucosal route. At 6 weeks after vaccination, mice were then challenged intranasally with 3.0 × 10 6 CFU of M. tuberculosis H37Ra in 100 μl PBS. At 14 weeks after immunization, the mice were sacrificed, the humoral and lung mucosal immune responses, cellular immune responses, and bacterial load were measured in murine lung and/or spleen. B , sera of BCG or rBCG PPE27 immunized mice were collected for antibodies detection using ELISA. C , the ratio of IgG2a/IgG1 in panel B was calculated. D , the levels of sIgA in BALF were assayed by ELISA. E , levels of the pro-inflammatory cytokines TNF-α and IFN-γ in mouse lungs and spleens determined by ELISA ( n = 3). F , bacterial loads in the lungs and spleens of immunized mice after M. tuberculosis H37Ra intranasal infection ( n = 6), respectively. The data represent log10 H37Ra CFU cultured per g of tissue homogenate. Data presented as mean ± SEM. Significant differences among groups compared to PBS, BCG, or rBCG PPE27 , ∗ p < 0.05, were determined using one-way ANOVA.

Article Snippet: Rapamycin-insensitive companion of mTOR ( RICTOR) fl/fl mice, HK1 fl/fl mice, LysM - Cre -expressing mice, and C57 black 6 (C57BL/6) mice were obtained from Cyagen Bio Co, Ltd (Shanghai, China).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Cell Culture