codon optimized catalytically inactive phgdh d175n, r236k, h283a  (Addgene inc)

Journal: bioRxiv

Article Title: Spatially Resolved Proteomic Profiling Uncovers Structural and Functional Regulators of the Axon Initial Segment

doi: 10.1101/2023.03.06.531334

Figure Lengend Snippet: (A) Representative images of DIV15 cortical neurons treated with 50 µM PHGDH inhibitor NCT503 or non-effective analogue Inac for 48 h. MAP2 labelling (blue) indicates healthy neuronal morphology with or without NCT503 treatment, while AnkG labelling (green) shows the AIS structural changes. Enlarged images of AnkG are presented in the right panel. Bar= 50 µm. (B) PHGDH inhibition by NCT503 for 48 h significantly decreases AnkG intensity in the AIS. Quantification of integrated AnkG intensity from experiments (A). Three times independent experiments were performed. Graphs are presented as mean ± SEM. Mann-Whitney test, ****p<0.0001. (C-D) PHGDH inhibition by 50 µM NCT503 for 48 h significantly decreases NF186 (C) and TRIM46 (D) intensity in the AIS. Two times independent experiments were performed. Graphs are presented as mean ± SEM. Unpaired t-test, ****p<0.0001. (E) Representative images of DIV21 cortical neurons treated with 50 µM PHGDH inhibitor CBR5884 or vehicle DMSO for 48 h. MAP2 labelling (blue) indicates healthy neuronal morphology with or without CBR5884 treatment, while AnkG labelling (green) shows the AIS structural changes. Enlarged images of AnkG are presented in the right panel. Bar= 50 µm. (F) PHGDH inhibition by CBR5884 for 48 h significantly decreases AnkG intensity in the AIS. Quantification of integrated AnkG intensity from experiments (E). Two times independent experiments were performed. Graphs are presented as mean ± SEM. Unpaired t-test, ****p<0.0001. (G-H) PHGDH inhibition by 50 µM CBR5884 for 48 h significantly decreases NF186 (G) and TRIM46 (H) intensity in the AIS. Two times independent experiments were performed. Graphs are presented as mean ± SEM. Mann-Whitney test for NF186 statistical analysis and Unpaired t-test for TRIM46 statistical analysis, ****p<0.0001. (I-J) PHGDH inhibition by NCT503 for 48 h treatment significantly shortens AIS length evaluated by AnkG (I) and NF186 (J). At least two times independent experiments were performed. Graphs are presented as mean ± SEM. Mann-Whitney test, ****p<0.0001; ***p<0.001. (K-L) PHGDH inhibition by CBR5884 for 48 h treatment significantly shortens AIS length evaluated by AnkG (K) and NF186 (L). Two times independent experiments were performed. Graphs are presented as mean ± SEM. Mann-Whitney test, ****p<0.0001; *p<0.05. (M-N) The effects of PHGDH inhibitor NCT503 on AIS is independent of micro-environmental astrocytes. Cortical neurons were cultured with astrocytes as feeder layer. AraC was added after 1 day plating to curb glial cells proliferation. The neuronal treatment in conditioned medium is defined as ‘–astrocytes’, while in the neuron-glia cultured system is defined as ‘+astrocytes’. Quantification of integrated AnkG intensity (M) and AIS length (N) in DIV14 cortical neurons after 48 h treatment with 50 µM NCT503 or Inac. Two times independent experiments were performed. Graphs are presented as mean ± SEM. Mann-Whitney test, ****p<0.0001. (O-P) The effects of PHGDH inhibitor CBR5884 on AIS with or without astrocytes. Quantification of integrated AnkG intensity (O) and AIS length (P) in DIV12 cortical neurons after 3 h treatment with 50 µM CBR5884 or DMSO. Two times independent experiments were performed. Graphs are presented as mean ± SEM. Mann-Whitney test, ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.

Article Snippet: PHGDH inhibitor NCT503 (GLPBIO, Cat# GC15943) and its control non-active analogue PHGDH-inactive (GLPBIO, Cat# GC17293) were dissolved in DMSO and stored with a concentration of 10 mM.

Techniques: Inhibition, MANN-WHITNEY, Cell Culture

Journal: bioRxiv

Article Title: Spatially Resolved Proteomic Profiling Uncovers Structural and Functional Regulators of the Axon Initial Segment

doi: 10.1101/2023.03.06.531334

Figure Lengend Snippet: (A-B) PHGDH inhibition by 25 and 50 µM NCT503 for 48 h reduces AnkG intensity in the AIS (A) and shortens AIS length (B). Data are normalized against 25 µM Inac samples. Two times independent experiments were performed. Graphs are presented as mean ± SEM. Statistical analysis was applied to AnkG intensity using Kruskal-Wallis test with Dunn’s correction and AIS length using Tukey’s multiple comparisons test. ****p<0.0001; ***p<0.001; **p<0.01; ns, not significant. (C-D) PHGDH inhibition by CBR5884 for 48 h treatment reduces AnkG intensity (C) and AIS length (D). DIV19 cortical neurons were treated with 30, 50 and 100 µM PHGDH inhibitor CBR5884 for 48 h. Data are normalized against vehicle. Graphs are presented as mean ± SEM. Statistical analysis was applied to AnkG intensity using Tukey’s multiple comparisons test and AIS length using Kruskal-Wallis test with Dunn’s correction. ****p<0.0001; **p<0.01; ns, not significant. (E) Representative images of cortical neurons showing NF186 and TRIM46 decrease after 48 h treatment with 50 µM PHGDH inhibitor NCT503 or CBR5884. Bar= 50 µm. (F-G) 3 h treatment with 50 µM NCT503 leads to significant reduction of AnkG intensity (F) and slight shortening of AIS length (G). DIV11-15 neurons were treated with 50 µM NCT503 or Inac. Data are normalized against control. Two times independent experiments were performed. Graphs are presented as mean ± SEM. Mann-Whitney test, *p<0.05. (H-I) 3 h treatment with 50 µM CBR5884 leads to significant reduction of AnkG intensity (H) and AIS length (I). DIV11-19 cortical neurons were treated with CBR5884 or vehicle DMSO. Three times independent experiments were performed. Graphs are presented as mean ± SEM. Mann-Whitney test, ****p<0.0001. (J) Workflow for recovery experiments after PHGDH inhibition. Cortical neurons were first treated with 50 µM NCT503 or Inac for 48 h, followed by twice washout and then maintained in conditioned medium for 7 h and 24 h individually for recovery. (K) Representative images of DIV14 neurons stained by AnkG antibody showing AIS structure recovery after 24 h maintenance in conditioned medium. (L-M) Quantification of integrated AnkG intensity (L) in the AIS or AIS length (M) after different recovery periods (0, 7 and 24 h). Data are normalized against the corresponding control in each time point. Two times independent experiments were performed. Graphs are presented as mean ± SEM. Statistical analysis was applied to AnkG intensity using Mann-Whitney test and AIS length using Unpaired t-test. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

Article Snippet: PHGDH inhibitor NCT503 (GLPBIO, Cat# GC15943) and its control non-active analogue PHGDH-inactive (GLPBIO, Cat# GC17293) were dissolved in DMSO and stored with a concentration of 10 mM.

Techniques: Inhibition, Control, MANN-WHITNEY, Staining