Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: YY2 protein expression in Tsc2−/− 105K cells ( a ) and Tsc2 KO MEFs ( b ) treated with 20 nM rapamycin (Rapa) or vehicle for 24 hr ( n = 3). YY2 protein expression in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ) treated with 10 μM PF-4708671 (S6K1 inhibitor) or vehicle for 24 hr ( n = 3). e Immunoblot analysis of HeLa cells transfected with eGFP-YY2, wild-type S6K (HA-WT-S6K), or kinase-dead S6K (HA-KD-S6K) for 48 hr ( n = 3). f Immunoblot analysis of in vitro kinase assays of recombinant active S6K in the presence or absence of myc-YY2 as a substrate. eIF4B was used as a positive control ( n = 3). g Immunoblot analysis of immunoprecipitated (IP) GFP-YY2 and the whole-cell-lysate (WCL) from HeLa cells expressing GFP-YY2 ( n = 3). h Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing GFP-YY2 ( n = 3). i The evolutionarily conserved putative S6K phosphorylation site in YY2. j Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells stably expressing wild-type (WT) GFP-YY2 or mutant (T336A) GFP-YY2, treated with 40 μM PF-4708671 for 1 hr ( n = 3). k Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing WT GFP-YY2 or T336A GFP-YY2 ( n = 3). l , m Network view of predicted E3 ubiquitin ligase-YY2 interactions by UbiBrowser 2.0. Confidence level of ( m ). n Decreased EGFP-YY2 expression in HeLa cells transfected with Smurf1-FLAG ( n = 3). o Decreased Smurf1 ubiquitination in HeLa cells transfected with S6K. Immunoblot analysis of GFP-immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG or HA-WT-S6K for 24 hr ( n = 3). p Immunoblot analysis of GFP-and IgG control immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG ( n = 3). q Immunoblot analysis of B7-H3 expression in Tsc2−/− 105K cells, transfected with control or myc-YY2 for 48 hr and treated with 20 nM rapamycin or vehicle for the last 24 hr ( n = 3). Source data is provided in the Source data file.

Article Snippet: HeLa cells stably expressing wild-type eGFP-YY2 or mutant T336A eGFP-YY2 were treated with 30 µM PF-4708971 or transfected with plasmids expressing WT-HA-S6K and Smurf1-FLAG for 24 hr, and then treated with MG132 at 20 µM for 5 hr before cell lysis with 1 x RIPA buffer (CST, Cat#9806S).

Techniques: Expressing, Western Blot, Transfection, In Vitro, Recombinant, Positive Control, Immunoprecipitation, Stable Transfection, Mutagenesis

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: YY2 protein expression in Tsc2−/− 105K cells ( a ) and Tsc2 KO MEFs ( b ) treated with 20 nM rapamycin (Rapa) or vehicle for 24 hr ( n = 3). YY2 protein expression in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ) treated with 10 μM PF-4708671 (S6K1 inhibitor) or vehicle for 24 hr ( n = 3). e Immunoblot analysis of HeLa cells transfected with eGFP-YY2, wild-type S6K (HA-WT-S6K), or kinase-dead S6K (HA-KD-S6K) for 48 hr ( n = 3). f Immunoblot analysis of in vitro kinase assays of recombinant active S6K in the presence or absence of myc-YY2 as a substrate. eIF4B was used as a positive control ( n = 3). g Immunoblot analysis of immunoprecipitated (IP) GFP-YY2 and the whole-cell-lysate (WCL) from HeLa cells expressing GFP-YY2 ( n = 3). h Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing GFP-YY2 ( n = 3). i The evolutionarily conserved putative S6K phosphorylation site in YY2. j Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells stably expressing wild-type (WT) GFP-YY2 or mutant (T336A) GFP-YY2, treated with 40 μM PF-4708671 for 1 hr ( n = 3). k Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing WT GFP-YY2 or T336A GFP-YY2 ( n = 3). l , m Network view of predicted E3 ubiquitin ligase-YY2 interactions by UbiBrowser 2.0. Confidence level of ( m ). n Decreased EGFP-YY2 expression in HeLa cells transfected with Smurf1-FLAG ( n = 3). o Decreased Smurf1 ubiquitination in HeLa cells transfected with S6K. Immunoblot analysis of GFP-immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG or HA-WT-S6K for 24 hr ( n = 3). p Immunoblot analysis of GFP-and IgG control immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG ( n = 3). q Immunoblot analysis of B7-H3 expression in Tsc2−/− 105K cells, transfected with control or myc-YY2 for 48 hr and treated with 20 nM rapamycin or vehicle for the last 24 hr ( n = 3). Source data is provided in the Source data file.

Article Snippet: HeLa cells stably expressing wild-type eGFP-YY2 or mutant T336A eGFP-YY2 were treated with 30 µM PF-4708971 or transfected with plasmids expressing WT-HA-S6K and Smurf1-FLAG for 24 hr, and then treated with MG132 at 20 µM for 5 hr before cell lysis with 1 x RIPA buffer (CST, Cat#9806S).

Techniques: Expressing, Western Blot, Transfection, In Vitro, Recombinant, Positive Control, Immunoprecipitation, Stable Transfection, Mutagenesis

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: a Knockdown of B7-H3 using two different shRNAs in Tsc2−/− 105K cells inhibits subcutaneous tumor growth in syngeneic wild-type (WT) C57BL/6 J mice compared with non-targeting shRNA (sh-NC). b Growth of subcutaneous sh-NC, sh-B7-H3 (1), and sh-B7-H3 (2) Tsc2−/− 105K tumors in WT C57BL/6 J mice. n = 8 mice/group, means ± SD, one-way ANOVA with Holm-Sidak’s multiple comparisons test, **** p < 0.0001. c Tumor-free survival curve of mice in b . Log-rank analysis. d Growth of subcutaneous sh-NC or sh-B7-H3 (1) Tsc2−/− 105K tumors in WT or Rag1 −/− C57BL/6 J mice. n = 5 mice/group, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01 **** p < 0.0001. e Tumor volume of d 35-day post cell inoculation. n = 5 mice/group, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01 **** p < 0.0001. f Density viSNE plots from CyTOF data on pre-gated CD45 + tumor-infiltrating lymphocytes (TILs) from sh-NC or sh-B7-H3 (1) Tsc2−/− 105K tumors. g Percentage of indicated cell types within CD45 + TILs in f . n = 6 or 8 tumors for sh-NC group, n = 4 or 5 tumors for sh-B7-H3 (1), n = 4, 5, or 6 tumors for sh-B7-H3 (2). Exact n is indicated in Source data file. Means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. h Top panel: Representative images of CD3, CD4 and CD8 IHC staining on tumor sections from each group. Scale bar = 100 μm. i Quantification of CD3 + , CD4 + and CD8 + T cells in each group ( n = 9/group). Means ± SD, one-way ANOVA with Dunnett’s multiple comparisons test, **** p < 0.0001. Source data and exact p values are provided in the Source data file.

Article Snippet: HeLa cells stably expressing wild-type eGFP-YY2 or mutant T336A eGFP-YY2 were treated with 30 µM PF-4708971 or transfected with plasmids expressing WT-HA-S6K and Smurf1-FLAG for 24 hr, and then treated with MG132 at 20 µM for 5 hr before cell lysis with 1 x RIPA buffer (CST, Cat#9806S).

Techniques: shRNA, Immunohistochemistry

Journal: Nature Communications

Article Title: Microbiota-derived acetate enhances host antiviral response via NLRP3

doi: 10.1038/s41467-023-36323-4

Figure Lengend Snippet: a – c WT and Nlrp3 −/− mice were given 50 mM SA in drinking water and then intranasally infected with PR8 as depicted in Fig. : relative Ifna1 expression to Gapdh ( n = 4, n = 4) ( a ) and viral load in lung homogenates ( n = 5, n = 5) ( b ) on day 7 post-infection and weight loss ( n = 10, n = 12) ( c ) were assessed. d – e WT and Nlrp3 −/− BMDMs were pretreated with 250 μM SA for 24 h and then transfected with 200 ng polyI:C for 12 h ( d ) ( n = 2) or 1 h ( e ). d Concentrations of IFN-α2/4 released from BMDMs. e Crude mitochondria extracts were subjected to SDD-AGE and immunoblotted with anti-MAVS antibody, and the intensity of MAVS aggregation was analyzed with ImageJ v2.0 and marked below. The cell lysates were subjected to immunoblot analysis. The relative ratios of pIRF3 (Ser396) to total IRF3 were analyzed with ImageJ v2.0 and marked below. f , h , j Western blot analysis of co-immunoprecipitation of NLRP3 with MAVS or GPR43 from cell lysates of HEK293T cells transfected with plasmids expressing V5-tagged NLRP3 and FLAG-tagged MAVS ( f ) or FLAG-tagged NLRP3 and V5-tagged GPR43 ( h ) or V5-tagged GPR43, eGFP-tagged NLRP3 and FLAG-tagged MAVS ( j ). g , i PRP-eGFP-mNlrp3-retrovirus infected BMDMs were pretreated with 250 μM SA for 24 h and then transfected with 200 ng polyI:C for 1 h. Fluorescence images of eGFP-mNLRP3 (green), MAVS (red) or GPR43 (red) and cell nuclei (blue) were captured with confocal microscope. k Western blot analysis of sequential IP in overexpressing systems. The mutual interaction among NLRP3, MAVS and GPR43 were confirmed. The black arrow indicates the specific FLAG-MAVS band. Results represent two ( a – c , g , i , k ) or three independent experiments ( d – f , j ). Data in ( a – d ) are presented as mean (± SEM), two-tailed Student’s t test ( a – c) , one-way ANOVA with Dunnett’s post-hoc test ( d ). Significant values are defined by * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: For NLRP3 colocalization with MAVS or GPR43, Nlrp3 −/− BMDMs were infected with PRP-eGFP-mNlrp3 retrovirus and pretreated with or without 250 μM sodium acetate for 24 h and then transfected with 200 ng polyI:C for 1 h. The cells were fixed, blocked, and then incubated with rabbit anti-MAVS (Cat# 4983, rodent specific, CST) or rabbit anti-GPR43 (bs-13536R, Bioss) for 12 h at 4 °C, goat-anti-rabbit IgG (AF555 conjugated) for 2 h at room temperature and DAPI for 1 min at room temperature.

Techniques: Infection, Expressing, Transfection, Western Blot, Immunoprecipitation, Fluorescence, Microscopy, Two Tailed Test

Journal: Nanoscale Advances

Article Title: Scavenging neurotoxic aldehydes using lysine carbon dots

doi: 10.1039/d2na00804a

Figure Lengend Snippet: Toxicity of DOPAL and the protective effect of Lys C-dots in the neuronal SH-SY5Y cells were studied by XTT assay. The left side shows the viability of the cells in the medium (grey histogram), in the presence of 100 μM DOPAL (black histogram), and in the presence of 1 mg mL −1 Lys C-dots (blue histogram); the right section shows the effect of increasing concentrations of Lys-C-dots on cell viability in the presence of 100 μM DOPAL (purple histograms).

Article Snippet: After treatment, α-Syn-EGFP expressing SH-SY5Y cells were harvested in RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Roche).

Techniques: XTT Assay

Journal: Nanoscale Advances

Article Title: Scavenging neurotoxic aldehydes using lysine carbon dots

doi: 10.1039/d2na00804a

Figure Lengend Snippet: Protective effect of Lys C-dots against DOPAL-induced cytotoxicity in SH-SY5Y cells. (A) Cell morphology analysis by Giemsa staining. Representative bright field images and relative high magnification images (inset) are shown. DOPAL toxicity results in accumulation of rounded cells (black outlined white arrowheads) per unit area, whereas pre-treatment with 2.5 mg mL −1 Lys C-dots reduced the DOPAL effect. (B) Quantification of the number of rounded cells per unit area (μm 2 ) was performed in n = 10 images for each condition and data are showed as mean ± SEM. Scale bar: 50 μm.

Article Snippet: After treatment, α-Syn-EGFP expressing SH-SY5Y cells were harvested in RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Roche).

Techniques: Staining

Journal: Nanoscale Advances

Article Title: Scavenging neurotoxic aldehydes using lysine carbon dots

doi: 10.1039/d2na00804a

Figure Lengend Snippet: Validation of the SH-SY5Y stable cell line over-expressing α-Syn-EGFP and recovering of the DOPAL effect by treatment with Lys C-dots. (A) Immunofluorescence of SH-SY5Y stable cells overexpressing α-Syn-EGFP (green) and stained with an anti-α-Syn (MJFR1) antibody (red) to display co-localization. Nuclei are stained with Hoechst (blue), scale bar 50 μM. (B) Western blot of SH-SY5Y cells over-expressing α-Syn-EGFP treated with 100–200 μM DOPAL at different time points (18–24 hours). DOPAL induced dimers revealed by the anti-α-Syn (MJFR1) antibody are detected at 90–100 kDa. (C) Western blot analysis of lysates of SH-SY5Y cells over-expressing α-Syn-EGFP pre-treated with specified concentrations of Lys C-dots for 24 hours, following 150 μM DOPAL treatment for 18 hours. (D) In the quantification, the band intensity of the α-Syn-EGFP dimer was normalized to HSP70. Data are presented as mean ± SEM ( n = 3).

Article Snippet: After treatment, α-Syn-EGFP expressing SH-SY5Y cells were harvested in RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Roche).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Staining, Western Blot