Fig 1: Pathological Analyses of LRRK2-G2019S Knockin 3D Organoids(A) Representative images showing colocalization analysis of EEA1-positive endosomes with pS129-a-synuclein-positive puncta in wild-type and LRRK2-G2019S 3D organoids. Scale bar, 20 µm.(B) Percentage of pS129-a-synuclein-positive and EEA1-positive puncta among all EEA1-positive puncta (left). Number of pS129-a-synuclein/EEA1-positive endosomes per cell in LRRK2-G2019S 3D organoids (right). Data represent the mean ± SEM. *p < 0.05, **p < 0.01 by ANOVA (n = 10 per sample).(C) Thioflavin T (50 µM) staining of wild-type and LRRK2-G2019S 3D organoids. The arrow indicates a-synuclein deposits. Scale bars, 20 µm.(D) Percentage of thioflavin T-positive cells among all TH-positive cells in the wild-type and LRRK2-G2019S 3D organoids. Data represent the mean ± SEM. **p < 0.01 by ANOVA (n = 3 per sample).(E) Quantification of the mean area of thioflavin T-positive deposits in midbrain 3D organoids at 60 days. Data represent the mean ± SEM. **p < 0.01 by ANOVA (n = 5 per sample).(F and G) (F) Treatment with the LRRK2 kinase inhibitor (GSK2578215A, 1 µM) significantly reduces phosphorylated a-synuclein oligomer levels in LRRK2-G2019S 3D organoids. (G) Intensity of pS129-a-synuclein in LRRK2-G2019S 3D organoids treated with the LRRK2 kinase inhibitor. Data represent the mean ± SEM. *p < 0.05 by ANOVA (n = 3 per sample).(H) Real-time qPCR analysis of dopaminergic neuron markers (TH, AADC, and DAT) in wild-type and LRRK2-G2019S 3D organoids treated with the LRRK2 kinase inhibitor GSK2578215A. Data represent the mean ± SEM. *p < 0.05 by ANOVA (n = 3 per sample).
Fig 2: Generation of Midbrain 3D Organoids from LRRK2-G2019S hiPSCs(A) Immunofluorescence staining of midbrain 3D organoids from LRRK2-G2019S hiPSCs. Scale bars, 100 µm.(B) qRT-PCR analysis of midbrain 3D organoids and LRRK2-G2019S 3D organoids regarding dopaminergic neuronal markers TH, AADC, and DAT at 60 days. Data represent the mean ± SEM. *p < 0.05, **p < 0.01 by ANOVA (n = 3 per sample).(C) Immunostaining of TH-positive and cleaved caspase-3-positive cells in midbrain 3D organoids and LRRK2-G2019S 3D organoids. Scale bars, 20 µm.(D) Percentage of cleaved caspase-3/TH-positive cells in midbrain 3D organoids and LRRK2-G2019S 3D organoids treated with 0.5 mM MPTP. Data represent the mean ± SEM. *p < 0.05, **p < 0.01 by ANOVA (n = 3 per sample).(E and F) Western blot analysis (E) and quantification (F) show an increase in cleaved caspase-3 levels after treatment with MPTP. Data represent the mean ± SEM. *p < 0.05, **p < 0.01 by ANOVA.
Fig 3: Functional enrichment analysis of DEGs between DDC subgroups. (A) The volcano plot of DEGs between DDC subgroups in KIRC. (B) The KEGG pathways and GO functional enrichment analysis of DEGs between DDC subgroups. (C) The Spearman correlation between DDC expression and tumor proliferation signature (R = −0.15; P <.001). DDC, L-dopa decarboxylase; KIRC, Kidney renal clear cell carcinoma; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Fig 4: Identification of DDC expression in regulating amino acid metabolism of ccRCC. (A) The FUSCC proteomic ccRCC cohort demonstrates that amino acid metabolism is extensively dysregulated between tumor and normal tissue and the DDC protein is significantly downregulated in ccRCC. (B) Representative immunohistochemical (IHC) staining of DDC protein in normal kidney and ccRCC tissues. (C) Proteomic cohorts (FUSCC and CPTAC) showed DDC protein is lower in tumors than in normal tissue. (D) Transcriptomic cohorts (GSE36859, GSE40435, and GSE53757) showed DDC mRNA is lower in tumors than in normal tissue. (E) Pan-cancer analysis of DDC mRNA expression in human cancers. CPTAC, Clinical Proteomic Tumor Analysis Consortium; ccRCC, clear cell renal cell carcinoma; DDC, L-dopa decarboxylase; mRNA, messenger RNA (**P <.01; ***P <.001; ****P <.0001).
Fig 5: DDC expression correlated with immune microenvironment regulation in ccRCC. (A) The differences in immune cell infiltration between DDC subgroups. (B) The differences of ICP genes between DDC subgroups. (C) The differences in TIDE scores between DDC subgroups. The TIDE score is significantly higher in the DDCLow group (P <.0001). (D) The pan-cancer correlations between immune cell infiltrations and DDC expression. DDC, L-dopa decarboxylase; ICP, immune checkpoint; TIDE, Tumor Immune Dysfunction and Exclusion (*P <.05; **P <.01; ***P <.001; ****P <.0001).
Supplier Page from Abcam for Anti-DOPA Decarboxylase/DDC antibody [CL2962]