Journal: CNS Neuroscience & Therapeutics

Article Title: ICA1 affects APP processing through the PICK1‐PKCα signaling pathway

doi: 10.1111/cns.14754

Figure Lengend Snippet: ICA1 affects APP processing through PICK1‐PKCα signaling pathway. (A) Western blotting showed that ICA1 affects APP processing via the PICK1‐PKCα signaling pathway. (B) Quantification of the relative protein level of ICA1 ( n = 3) in the ICA1 overexpression group compared to the vector group in 20E2, * p < 0.05 for both. The data conformed to a normal distribution by Shapiro‐Wilk test. p Value was determined by a two‐tailed Student's t ‐test. (C) Quantification of the relative protein level of PICK1 ( n = 3) in the ICA1 overexpression group compared with that in the vector group in 20E2 cells, p > 0.05. The data conformed to a normal distribution by Shapiro‐Wilk test. p Value was determined by a one‐way ANOVA test. (D) Quantification of the relative protein level of PKCα ( n = 3) after ICA1 overexpression and PKCα inhibition, * p < 0.05. (E) Quantification of the relative protein level of p‐PKCα ( n = 3) after ICA1 overexpression and PKCα inhibition, ** p < 0.01, *** p < 0.001. (F) Quantification of the relative protein level of C83 ( n = 3) after ICA1 overexpression and PKCα inhibition, * p < 0.05, ** p < 0.01, *** p < 0.001. (G) Quantification of the relative protein level of APP ( n = 3) after ICA1 overexpression and PKCα inhibition, * p < 0.05, ** p < 0.01, *** p < 0.001. (H) Quantification of the relative protein level of ADAM10 ( n = 3) after ICA1 overexpression and PKCα inhibition, * p < 0.05, *** p < 0.001. (I) Quantification of the relative protein level of ADAM17 ( n = 3) after ICA1 overexpression and PKCα inhibition, * p < 0.05, ** p < 0.01.The data for each group conformed to a normal distribution by Shapiro‐Wilk test. p Value was determined by a one‐way ANOVA test.

Article Snippet: The Gapdh (60004–1‐1 g, 1:200, 000) and PICK1 (A1519, 1:1, 000) antibodies were purchased from Proteintech and ABclonal, respectively.

Techniques: Western Blot, Over Expression, Plasmid Preparation, Two Tailed Test, Inhibition

Journal: Nature Communications

Article Title: Nanoparticle-mediated TRPV1 channel blockade amplifies cancer thermo-immunotherapy via heat shock factor 1 modulation

doi: 10.1038/s41467-023-38128-x

Figure Lengend Snippet: CLSM images of HSF1 (green) ( a ) and corresponding co-localization rate analysis of HSF1 with nucleus (blue) ( b ) in PANC02 tumor cells treated with I-Micelles or IS-Micelles under light irradiation (+) or not ( n = 3 independent experiments). Scale bars, 50 μm. CLSM images of TGFβ1 (green) ( c ) and corresponding fluorescence intensity analysis ( d ) of PANC02 tumor sections from the mice treated with different formulations ( n = 3 independent experiments). Scale bars, 50 μm. CLSM images of α-SMA (green) ( e ) and corresponding fluorescence intensity analysis ( f ) of PANC02 tumor sections from the mice treated with different formulations ( n = 3 independent experiments). Scale bars, 50 μm. Masson’s trichrome staining (MTS) ( g ) and corresponding fibrosis analysis ( h ) of PANC02 tumor sections from the mice treated with different formulations ( n = 3 independent experiments). Scale bars, 50 μm. i CLSM images of HSF1 (green) translocation in PANC02 tumor cells treated with IS-Micelles and HSF1A under light irradiation (+), and CLSM images of TGFβ1 (green), α-SMA (green) and Masson’s trichrome staining (MTS) of PANC02 tumor sections from the mice treated with IS-Micelles and HSF1A under light irradiation (+) ( n = 3 independent experiments). Scale bars, 50 μm. CLSM images of Cy5 labeled aPD-L1 (red) ( j ) and corresponding fluorescence intensity analysis ( k ) in PANC02 tumor sections at 24 h post-injection of aPD-L1 from the mice treated with different formulations. Scale bars, 100 μm. This experiment was repeated three times independently with similar results. In c , e , g , and j , the mice were treated with PBS, IS-Micelles, I-Micelles under light irradiation (+), and IS-Micelles under light irradiation (+), respectively. Data are presented as mean ± SD ( b , d , f , h ). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.

Article Snippet: Then, the tissue sections with 10.0 μm thickness were made in a cryostat at 6 h post-irradiation or 24 h post-irradiation or 72 h post-injection, followed by the hematoxylin and eosin (H&E) staining, Ki67 staining (Abcam, ab15580, dilution: 1:100) TUNEL assay (green or red), Masson’s trichrome staining, TGFβ1 staining (Abclonal, A15103, dilution: 1:100), α-SMA staining (Abclonal, A2319, dilution: 1:100), FAPα staining (Abclonal, A6349, dilution: 1:100), collagen I staining (Abclonal, A5786, dilution: 1:100), fibronectin staining (Abclonal, A16678, dilution: 1:100), VEGF-A staining (Abcam, ab52917, dilution: 1:100), and HSP70 staining (Abcam, ab181606, dilution: 1:100) and further labeled with Alexa 594 labeled goat anti-rabbit IgG H&L secondary antibody (Abcam, ab150080, dilution: 1:200) or Alexa 488 labeled goat anti-rabbit IgG H&L secondary antibody (Abcam, ab150077, dilution: 1:200) for 0.5 h in different tissues according to the manufacturer’s protocols.

Techniques: Irradiation, Fluorescence, Staining, Translocation Assay, Labeling, Injection

Journal: Nature Communications

Article Title: Nanoparticle-mediated TRPV1 channel blockade amplifies cancer thermo-immunotherapy via heat shock factor 1 modulation

doi: 10.1038/s41467-023-38128-x

Figure Lengend Snippet: a Timeline for the treatment of subcutaneous PANC02 tumor model. Tumor growth profiles ( b ) and survival curves ( c ) of the mice bearing large PANC02 tumor model (250–300 mm 3 ) treated with various formulations ( n = 7 mice per group). Quantification of CD45 + CD11b + Gr−1 + MDSCs ( d ), CD11b + F4/80 + CD206 + TAMs ( e ) and CD11b + F4/80 + CD80 + TAMs ( f ) inside PANC02 tumors at 72 h post-treatment with various formulations ( n = 5 mice per group). g Ratio of CD11b + F4/80 + CD80 + TAMs to CD11b + F4/80 + CD206 + TAMs inside large PANC02 tumors at 72 h post-treatment with various formulations ( n = 5 mice per group). h Quantification of matured dendritic cells (CD11c + CD80 + CD86 + DCs) inside tumor-draining lymph nodes at 72 h post-treatment with various formulations ( n = 5 mice per group). Quantification of tumor-infiltrating CD45 + CD3 + CD8 + CTLs ( i ) and CD45 + CD3 - CD335 + NK cells ( j ) in PANC02 tumors at 72 h post-treatment with various formulations ( n = 5 mice per group). k Schematic illustration of ECM remodeling to facilitate aPD-L1 infiltration for inducing durable immune response via TRPV1 blockade-synergized thermotherapy. Upon TRPV1 blockade, nuclear translocation of HSF1 is effectively blocked which subsequently suppresses TGFβ1 upregulation and secretion, thus leading to inhibition of CAFs proliferation and activation-mediated ECM proteins deposition, ultimately promoting the infiltration of aPD-L1 and immune cells (e.g. NK cells and T lymphocytes) for durable immune response. Dash lines indicate the failure of downstream signals transduction upon blockade of hyperthermia-activated TRPV1 channel. The mice in b–j were treated with PBS (G1), aPD-L1 (G2), Abraxane/GEM (G3), I-Micelles under light irradiation (+) (G4), I-Micelles/aPD-L1 under light irradiation (+) (G5), IS-Micelles (G6), IS-Micelles/aPD-L1 (G7), IS-Micelles under light irradiation (+) (G8), and IS-Micelles/aPD-L1 under light irradiation (+) (G9), respectively. Data are presented as mean ± SD ( b , d – j ). For b and d – j , statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. For c , statistical significance was calculated by log-rank test. Source data are provided as a Source Data file.

Article Snippet: Then, the tissue sections with 10.0 μm thickness were made in a cryostat at 6 h post-irradiation or 24 h post-irradiation or 72 h post-injection, followed by the hematoxylin and eosin (H&E) staining, Ki67 staining (Abcam, ab15580, dilution: 1:100) TUNEL assay (green or red), Masson’s trichrome staining, TGFβ1 staining (Abclonal, A15103, dilution: 1:100), α-SMA staining (Abclonal, A2319, dilution: 1:100), FAPα staining (Abclonal, A6349, dilution: 1:100), collagen I staining (Abclonal, A5786, dilution: 1:100), fibronectin staining (Abclonal, A16678, dilution: 1:100), VEGF-A staining (Abcam, ab52917, dilution: 1:100), and HSP70 staining (Abcam, ab181606, dilution: 1:100) and further labeled with Alexa 594 labeled goat anti-rabbit IgG H&L secondary antibody (Abcam, ab150080, dilution: 1:200) or Alexa 488 labeled goat anti-rabbit IgG H&L secondary antibody (Abcam, ab150077, dilution: 1:200) for 0.5 h in different tissues according to the manufacturer’s protocols.

Techniques: Translocation Assay, Inhibition, Activation Assay, Transduction, Irradiation

Journal: mSystems

Article Title: Inhibition of Antiviral Innate Immunity by Avibirnavirus VP3 via Blocking TBK1-TRAF3 Complex Formation and IRF3 Activation

doi: 10.1128/mSystems.00016-21

Figure Lengend Snippet: The CC1 domain of VP3 is required for downregulating IFN-β production by interacting with TRAF3. (A) Effects of VP3 on the components of the RLR signaling pathway. HEK293T cells were transfected with the IFN-β reporter, pRL-TK, and an empty vector, MDA5, MAVS, TBK1, or IRF3(5D) along with a control vector or a vector expressing VP3 for 36 h; the IFN-β activities were then measured using a dual-luciferase assay. (B) Identification of the interaction of VP3 with RLR pathway molecules. Flag-tagged MDA5, MAVS, TBK1, IRF3, or TRAF3 and Myc-VP3 were cotransfected into HEK293T cells for 48 h. The lysates were subjected to a Co-IP assay with anti-Flag mouse MAb and immunoblotting with anti-Flag and anti-Myc rabbit PAb. (C) Endogenous TRAF3 colocalization with VP3 during IBDV infection. DF-1 cells were infected with IBDV and were subjected to immunofluorescence staining with anti-TRAF3 rabbit PAb and anti-VP3 mouse MAb. (D and E) Avibirnavirus VP3 interacts with endogenous TRAF3 during IBDV infection. HEK293T cells were infected with IBDV (MOI = 10) for 12 h, and cellular lysates were subjected to a Co-IP assay with anti-VP3 mouse MAb (D) or anti-TRAF3 rabbit PAb (E). Anti-IgG mouse or anti-IgG rabbit MAb served as a control. (F) VP3 interacts directly with TRAF3. Purified Flag-TRAF3 was separately mixed with the GST and GST-VP3 proteins, and a GST pulldown assay was conducted as described in Materials and Methods. (G) The CC1 domain of VP3 is responsible for the interaction with TRAF3. HEK293T cells were cotransfected with Myc-tagged VP3, VP3ΔCC1, VP3ΔCC2, or VP3ΔCC3 and Flag-TRAF3. Cellular lysates were subjected to a Co-IP assay with anti-Flag mouse MAb and Western blotting with anti-Flag and anti-Myc rabbit PAbs. (H) The CC1 domain is necessary for VP3 to downregulate IFN-β activation driven by MDA5. HEK293T cells were transfected with the IFN-β reporter, pRL-TK, an empty vector, and an expression plasmid encoding VP3 or VP3△CC1 along with MDA5 for 36 h; luciferase activity was then measured by a dual-luciferase assay.

Article Snippet: After being blocked with 5% skim milk, cells were incubated with anti-VP3 mouse MAb and anti-TRAF3 rabbit PAb (A15106; ABclonal Technology, Wuhan, China) overnight at 4°C.

Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Co-Immunoprecipitation Assay, Western Blot, Infection, Immunofluorescence, Staining, Purification, GST Pulldown Assay, Activation Assay, Activity Assay

Journal: mSystems

Article Title: Inhibition of Antiviral Innate Immunity by Avibirnavirus VP3 via Blocking TBK1-TRAF3 Complex Formation and IRF3 Activation

doi: 10.1128/mSystems.00016-21

Figure Lengend Snippet: The residue lysine-155 of TRAF3 is necessary for VP3 to downregulate IFN-β production. (A) Schematic representation of different domains of TRAF3 and its deletion mutants. aa, amino acids. (B) Identification of the TRAF3 domain binding to Avibirnavirus VP3. HEK293T cells were cotransfected with VP3 and Flag-tagged TRAF3, TRAF3△RING, TRAF3△zinc finger, or TRAF3△TRAF-C for 48 h. Cellular lysates were subjected to a Co-IP assay with anti-Flag mouse MAb and an immunoblotting assay using anti-Flag and anti-Myc rabbit PAbs. (C) The zinc finger domain of TRAF3 is essential for the interaction with VP3. A zinc finger fragment was cloned into the eGFP-C3 vector, and then the resulting construct and vector encoding Avibirnavirus VP3 were cotransfected into HEK293T cells for 48 h. Cellular lysates were subjected to a Co-IP assay with anti-VP3 mouse MAb and an immunoblotting assay with anti-GFP and anti-VP3 mouse MAbs. (D) Residual sequence of the zinc finger domain of TRAF3. The lysine residues are displayed in boldface. (E) The residue lysine-155 of TRAF3 is crucial for the interaction with VP3. Different Flag-TRAF3 mutants bearing a single lysine (K)-to-arginine (R) substitution and Myc-VP3 were cotransfected into HEK293T cells for 48 h. Cellular lysates were subjected to a Co-IP assay using anti-Flag mouse MAb and an immunoblotting assay with anti-Flag and anti-Myc rabbit PAbs. (F) TRAF3 -deficient HEK293T cells were generated by the CRISPR-Cas9 method. (G) Effects of the residue lysine-155 of TRAF3 on the inhibition of MDA5-mediated IFN-β activation by VP3. TRAF3 -deficient HEK293T cells were transfected with the IFN-β reporter and pRL-TK, along with an empty vector or a vector expressing VP3 together with MDA5 or MDA5 and TRAF3-WT or TRAF3-K155R for 36 h. The luciferase activities were assessed by a dual-luciferase assay. All data are presented as the means ± SD from three independent experiments. ns, P > 0.05; **, P < 0.01.

Article Snippet: After being blocked with 5% skim milk, cells were incubated with anti-VP3 mouse MAb and anti-TRAF3 rabbit PAb (A15106; ABclonal Technology, Wuhan, China) overnight at 4°C.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Clone Assay, Plasmid Preparation, Construct, Sequencing, Generated, CRISPR, Inhibition, Activation Assay, Transfection, Expressing, Luciferase

Journal: mSystems

Article Title: Inhibition of Antiviral Innate Immunity by Avibirnavirus VP3 via Blocking TBK1-TRAF3 Complex Formation and IRF3 Activation

doi: 10.1128/mSystems.00016-21

Figure Lengend Snippet: VP3 decreases the interaction of TRAF3 with TBK1 by inhibiting K33-linked polyubiquitination of the residue lysine-155 of TRAF3. (A) IBDV infection suppresses the ubiquitination of TRAF3. HEK293T cells were cotransfected with Flag-TRAF3 and HA-Ub or the corresponding empty vector for 24 h. The resultant cells were infected with IBDV for another 24 h. The lysates were subjected to immunoprecipitation and Western blotting assays with the indicated antibodies. (B) Viral protein VP3 represses TRAF3 ubiquitination. HEK293T cells were cotransfected with HA-Ub and Flag-TRAF3 along with an empty vector or Myc-VP3 for 48 h. Cellular lysates were subjected to a ubiquitination assay with anti-Flag mouse MAb and an immunoblotting assay using anti-HA mouse MAb and anti-Flag and anti-Myc rabbit PAbs. (C) Identification of the ubiquitination phenotype of the residue lysine-155 of TRAF3. Flag-TRAF3-WT or Flag-TRAF3-K155R and different HA-Ub mutants were cotransfected into HEK293T cells for 48 h. Cell lysates were subjected to a ubiquitination assay with anti-Flag mouse MAb and a Western blotting assay using anti-Flag rabbit PAb and anti-HA mouse MAb. (D) K33-linked ubiquitin chains enhance the association of TRAF3 and VP3. HEK293T cells were cotransfected with Flag-TRAF3 and Myc-VP3 along with HA-UbK33 or HA-UbK33R for 48 h. Cellular lysates were subjected to Flag precipitation and an immunoblotting assay with anti-Flag and anti-Myc PAbs. (E) The association between TRAF3 and VP3 is not affected by K11-linked polyubiquitin chains. Flag-TRAF3, Myc-VP3, and HA-UbK11 or an empty vector were cotransfected into HEK293T cells for 48 h. Cellular lysates were immunoprecipitated with anti-Flag mouse MAb, followed by immunoblotting using the indicated antibodies. (F) Impact of VP3 overexpression on TRAF3 K11- and K33-linked polyubiquitination. HEK293T cells were cotransfected with Flag-TRAF3, HA-UbK11, or HA-UbK33 and Myc-VP3 or the corresponding empty vector for 48 h. The lysates were subjected to immunoprecipitation and Western blotting assays using the indicated antibodies. (G) K33-linked polyubiquitination of the residue lysine-155 of TRAF3 benefits the interaction with TBK1. Flag-TRAF3-WT or a Flag-TRAF3-K155R mutant and Myc-TBK1 with or without HA-UbK33 were separately cotransfected into HEK293T cells for 48 h. Cellular lysates were subjected to a Co-IP assay with anti-Flag mouse MAb and an immunoblotting assay with anti-Flag and anti-Myc rabbit PAbs. (H) VP3 reduces the interaction of TRAF3 and TBK1. HEK293T cells were cotransfected with Flag-TRAF3 and Myc-TBK1 along with an empty vector or a vector expressing VP3 for 48 h. Cellular lysates were subjected to a Co-IP assay using anti-Flag mouse MAb and a Western blotting assay using anti-Flag and anti-Myc rabbit PAbs. (I) IBDV infection suppresses TRAF3 K33-linked polyubiquitination. HEK293T cells were cotransfected with Flag-TRAF3 and HA-UbK33 or the corresponding empty vector for 24 h, followed by infection with IBDV for another 24 h. The lysates were subjected to immunoprecipitation and a Western blotting assay with the indicated antibodies. (J) VP3 knockdown greatly blocks the decrease of IBDV-induced TRAF3 polyubiquitination. HEK293T cells were cotransfected with Flag-TRAF3 and HA-Ub or HA-UbK33 for 24 h, followed by infection with IBDV. At 1 h after infection, the resultant cells were transfected with the RNAi sequence against VP3 for another 23 h. Cellular lysates were subjected to immunoprecipitation and immunoblotting assays using the indicated antibodies. (K) The CC1 domain is required for VP3 to decrease the association of TRAF3 with TBK1. Flag-TRAF3, Myc-TBK1, and HA-UbK33 along with an empty vector, an expression plasmid encoding VP3 or VP3△CC1, were cotransfected into HEK293T cells for 48 h, respectively. Cellular lysates were subjected to a Co-IP assay with anti-Flag mouse MAb and an immunoblotting assay with anti-Flag and anti-Myc rabbit PAbs.

Article Snippet: After being blocked with 5% skim milk, cells were incubated with anti-VP3 mouse MAb and anti-TRAF3 rabbit PAb (A15106; ABclonal Technology, Wuhan, China) overnight at 4°C.

Techniques: Infection, Plasmid Preparation, Immunoprecipitation, Western Blot, Ubiquitin Assay, Over Expression, Mutagenesis, Co-Immunoprecipitation Assay, Expressing, Transfection, Sequencing

Journal: mSystems

Article Title: Inhibition of Antiviral Innate Immunity by Avibirnavirus VP3 via Blocking TBK1-TRAF3 Complex Formation and IRF3 Activation

doi: 10.1128/mSystems.00016-21

Figure Lengend Snippet: Primers used for cloning and quantitative real-time PCR

Article Snippet: After being blocked with 5% skim milk, cells were incubated with anti-VP3 mouse MAb and anti-TRAF3 rabbit PAb (A15106; ABclonal Technology, Wuhan, China) overnight at 4°C.

Techniques: Clone Assay, Sequencing