Journal: International Journal of Molecular Sciences

Article Title: A Potent Antagonist of Smoothened in Hedgehog Signaling for Epilepsy

doi: 10.3390/ijms232314505

Figure Lengend Snippet: The expression of Gli1 stimulated with Shh-CM was suppressed by TT22. ( A ) Serum-starved NIH3T3 cells were treated with Shh-CM for 24 h, then analyzed for mRNA levels of Gli1. ( B , C ) Serum-starved NIH3T3 cells were induced by Shh-CM with DMSO (vehicle), with an indicated concentration of TT22, 1 μM LDE-225 (LDE-1) or 1 μM GDC-0449 (GDC-1) for 24 h. Cells were analyzed for mRNA levels ( B ) and the protein levels ( C ) of Gli1. ( D ) Quantitation of Gli1 protein levels normalized to Tubulin loading control measured the dose-dependent inhibition of the Hh pathway activity by TT22 upon Shh stimulation. All data are means ± SEM (Student’s t -test and one-way ANOVA). *** p < 0.001.

Article Snippet: Cell lysates were harvested and detected by Western blotting with the following primary antibodies: Gli1 (CST, 2534s, 1:1000, Boston, MA, USA); and Tubulin (CST, 3873 s, 1:2000).

Techniques: Expressing, Concentration Assay, Quantitation Assay, Control, Inhibition, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: A Potent Antagonist of Smoothened in Hedgehog Signaling for Epilepsy

doi: 10.3390/ijms232314505

Figure Lengend Snippet: The expression of Gli1 stimulated with SAG was suppressed by TT22. ( A ) Serum-starved NIH3T3 cells were treated with 100 nM SAG for 24 h, then analyzed for mRNA levels of Gli1. ( B , C ) Serum-starved NIH3T3 cells were induced by 10 −7 M (100 nM) SAG with DMSO (vehicle), with the indicated concentration of TT22, 1 μM LDE-225 (LDE-1) or 1 μM GDC-0449 (GDC-1) for 24 h. Cells were analyzed for mRNA levels ( B ) and the protein levels ( C ) of Gli1. ( D ) Quantitation of Gli1 protein levels normalized to Tubulin loading control measured the dose-dependent inhibition of the Hh pathway activity by TT22 upon SAG stimulation. All data are means ± SEM (Student’s t -test and one-way ANOVA). *** p < 0.001.

Article Snippet: Cell lysates were harvested and detected by Western blotting with the following primary antibodies: Gli1 (CST, 2534s, 1:1000, Boston, MA, USA); and Tubulin (CST, 3873 s, 1:2000).

Techniques: Expressing, Concentration Assay, Quantitation Assay, Control, Inhibition, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: A Potent Antagonist of Smoothened in Hedgehog Signaling for Epilepsy

doi: 10.3390/ijms232314505

Figure Lengend Snippet: Scheme of the inhibition of Smoothened antagonists on seizure-like activity. Antagonists TT22, GDC-0449, and LDE-225 can bind with a Smoothened receptor, repress its activity, and then inhibit the expression of a downstream Gli1 transcription factor, thus suppressing the abnormal seizure-like activity in hippocampal neurons.

Article Snippet: Cell lysates were harvested and detected by Western blotting with the following primary antibodies: Gli1 (CST, 2534s, 1:1000, Boston, MA, USA); and Tubulin (CST, 3873 s, 1:2000).

Techniques: Inhibition, Activity Assay, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: PARD3 drives tumorigenesis through activating Sonic Hedgehog signalling in tumour-initiating cells in liver cancer.

doi: 10.1186/s13046-024-02967-3

Figure Lengend Snippet: Fig. 4 SHH signalling activation was responsible for stemness maintenance in PARD3-overexpressing CD133+ TICs. (A) CD133+ and CD133− cells were sorted from PARD3-overexpressing Hepa1-6 cells and subjected to RNA-seq analysis. (B) PARD3 activation induced perturbation of gene expression in CD133+ cells compared with CD133− cells. (C) Significant signalling pathways activated in CD133+ PARD3-overexpressing cells, as revealed by functional enrichment analysis. D&E. SHH signalling activity in different subclusters of cancer cells, as determined by single-cell sequencing. F. GSEA results showing that SHH signalling was activated in CD133+ PARD3-overexpressing cells. G. Relative expression of Gli1, PTCH1 and SHH in CD133+ and CD133− PARD3- overexpressing cells. H. Gli1 protein expression was increased in CD133+ PARD3-overexpressing cells. I. Quantification of the CD133+ population within PARD3-overexpressing and wild-type Hepa1-6 cells treated with GANT58 (n = 3). J. In vitro limiting dilution assay showing the tumour sphere formation ability of PARD3 OE/wild-type CD133+ cells with or without GANT58 treatment. K. The interaction between aPKC and PARD3 was increased upon PARD3 overexpression, as determined by co-IP. L. The phosphorylation of aPKC was increased upon PARD3 overexpression. M. Gli1 binding to the Sox2 promoter was attenuated by ZIP. N. Quantification of the CD133+ population within PARD3-overexpressing Hepa1-6 cells with or without inhibition of aPKC signal ling by ZIP. O. In vitro tumour sphere formation ability of PARD3 OE/wild-type CD133+ cells treated with ZIP, as determined by an in vitro limiting dilution assay. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not statistically significant

Article Snippet: The primary antibodies against SOX2 (A0561, ABclonal), Gli1 (V812, Cell Signaling Technology), phospho-aPKC (9378, Cell Signaling Technology), PARD3 (11085-1-AP, Proteintech), and CD133 (A0818, ABclonal) were labelled with Opal 480, Opal 520, Opal 570, Opal 650 and Opal 780, respectively.

Techniques: Activation Assay, RNA Sequencing, Gene Expression, Functional Assay, Activity Assay, Sequencing, Expressing, In Vitro, Limiting Dilution Assay, Over Expression, Co-Immunoprecipitation Assay, Phospho-proteomics, Binding Assay, Inhibition

Journal: Journal of experimental & clinical cancer research : CR

Article Title: PARD3 drives tumorigenesis through activating Sonic Hedgehog signalling in tumour-initiating cells in liver cancer.

doi: 10.1186/s13046-024-02967-3

Figure Lengend Snippet: Fig. 5 Clinical significance of PARD3, as revealed by multiplex IHC of a tissue microarray. (A) KM survival curve showing the prognostic value of overall PARD3 and Gli1 expression and their expression in CD133 + cells and CD133- cells. (B) Patients harbouring a high density of CD133 + cells with high PARD3 expression had the worst prognosis relative to patients in the other subgroups. (C) The levels of PARD3 and phospho-aPKC were significantly higher in CD133 + cells than in CD133- cells. (D) Representative image showing that higher expression of PARD3 was associated with higher presence of CD133 + stem like cancer cells. (E) Correlation heatmap showing that the expression levels of PARD3/aPKC/Gli1/Sox2 were positively intercorrelated in CD133+ cells. (F) Consensus clustering tools were used to classify the 88 patients into two subtypes as determined by setting k to 2. (G) Patients in cluster 1 exhibited higher activation of PARD3/aPKC/Gli1/Sox2 in CD133+ cells, accompanied by less favourable clinicopathological features and (H) worse over all survival. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not statistically significant

Article Snippet: The primary antibodies against SOX2 (A0561, ABclonal), Gli1 (V812, Cell Signaling Technology), phospho-aPKC (9378, Cell Signaling Technology), PARD3 (11085-1-AP, Proteintech), and CD133 (A0818, ABclonal) were labelled with Opal 480, Opal 520, Opal 570, Opal 650 and Opal 780, respectively.

Techniques: Multiplex Assay, Microarray, Expressing, Activation Assay

Journal: Biology

Article Title: Enhancement of Antiviral T-Cell Responses by Vitamin C Suggests New Strategies to Improve Manufacturing of Virus-Specific T Cells for Adoptive Immunotherapy

doi: 10.3390/biology11040536

Figure Lengend Snippet: The yield of CMV-specific T cells using CSA is slightly increased upon supplementation with vitamin C. CSA was performed using pp65pp and the magnetically enriched cells were analyzed by flow cytometry. Cells are gated on viable CD3 + lymphocytes based on CD45 expression, scatter properties (SSC-A), 7-AAD staining and CD3 expression. Number of IFN-γ-expressing T cells after enrichment, without (control), with pp65pp (pp65pp), or with pp65pp and vitamin C supplementation (pp65pp + vitamin C) during CSA shown as ( a ) representative plots, ( b ) frequency of CD3 + IFN-γ + T cells and ( c ) absolute numbers of CD3 + IFN-γ + T cells ( n = 5). ( b , c ) Each point represents an individual donor and mean ± SD is shown. ( d ) Exemplary number of CD3 + IFN-γ + T cells of a 58-year-old male donor. Shown are absolute cell numbers after cell sorting, comparable to results in ( b ). Friedman’s test and post hoc Dunn’s test were used to determine statistical significance (in ( b , c ); * p < 0.05).

Article Snippet: All samples were analyzed by multicolor flow cytometry (FACS Canto II, FACSDiva V8.1.2 software, FlowJo_v10.7.1 software; all BD Biosciences).

Techniques: Flow Cytometry, Expressing, Staining, FACS

Journal: Biology

Article Title: Enhancement of Antiviral T-Cell Responses by Vitamin C Suggests New Strategies to Improve Manufacturing of Virus-Specific T Cells for Adoptive Immunotherapy

doi: 10.3390/biology11040536

Figure Lengend Snippet: Vitamin C alone does not affect the memory phenotype distribution yet increases the fraction of T EMRA after antigenic stimulation. After PBMC isolation, cells were incubated with (+vitamin C) or without vitamin C for 3 days. On day 3, part of the untreated and all treated cells were stimulated overnight with pp65pp. CD45RA and CD62L expression were measured by flow cytometry on days 0, 3, and 4. Cells are gated on viable CD8 + and CD4 + T cells based on scatter properties (FSC-A/SSC-A), 7-AAD staining as well as CD3, CD8 and CD4 expression. Representative contour plots show percentages of naïve T cells (T N ; CD45RA + CD62L + ), central memory cells (T CM ; CD45RA − CD62L + ), effector memory cells (T EM ; CD45RA − CD62L − ) and late effector memory cells (T EMRA ; CD45RA + CD62L − ) among CD8 + ( a , c ) and CD4 + ( b , d ) T-cell subsets. Data are shown as representative ( a , b ) and summarized results ( c , d ) from n = 5 donors. Bar graphs represent mean and SD.

Article Snippet: All samples were analyzed by multicolor flow cytometry (FACS Canto II, FACSDiva V8.1.2 software, FlowJo_v10.7.1 software; all BD Biosciences).

Techniques: Isolation, Incubation, Expressing, Flow Cytometry, Staining