Journal: International Journal of Biological Sciences

Article Title: Glycine Decarboxylase Regulates Renal Carcinoma Progression via Interferon Stimulated Gene Factor 3-Mediated Pathway

doi: 10.7150/ijbs.104458

Figure Lengend Snippet: Suppression of GLDC induces dNTPs depletion, resulting in ROS accumulation and leading to mitochondrial stress in RCC cells. (A) Quantification of dTTP, dATP, dCTP, and dGTP levels in ACHN and Caki-2 cells. CTL, control; shGLDC, knockdown of GLDC. Data are shown as the means ± SD ( n =3). (B) Representative micrographs of immunofluorescence staining for mitoSOX with 4',6-diamidino-2-phenylindole (DAPI) counterstaining. ACHN cells were treated with brequinar (Bre) 10 µM for 48 h. ImageJ is used for quantification of the fluorescence intensity. NT, non-treated cells. Data are shown as the means ± SD ( n =3). (C) Representative micrographs of immunofluorescence staining for mitoSOX with DAPI counterstaining in control (CTL) and GLDC knock-downed (shGLDC) cells. The quantifications of fluorescence intensity are on the right side. Data are shown as the means ± SD ( n =3). MitoSOX staining intensity in ACHN CTL and shGLDC cells was confirmed by FACS analysis. Data are shown as the means ± SD ( n =3). (D) Representative micrographs of immunofluorescence staining for mitochondria (anti-HSP60) and mitochondrial (mt) DNA nucleoids (anti-DNA). ImageJ was used to calculate the size of mtDNA nucleoids. Mito, mitochondria. p values were calculated using two-tailed unpaired Student t -test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Brequinar (Bre) sodium was purchased from Tocris, Bristol, UK.

Techniques: Control, Knockdown, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Glycine Decarboxylase Regulates Renal Carcinoma Progression via Interferon Stimulated Gene Factor 3-Mediated Pathway

doi: 10.7150/ijbs.104458

Figure Lengend Snippet: GLDC regulates RCC cell proliferation by activating ISGF3 through the inhibition of dNTP synthesis. (A) Growth curve of ACHN cells treated with brequinar (Bre) 10 µM or without (NT) was assessed by CCK-8 assays. Data are shown as the means ± SD ( n =3). (B) Western blot of indicated proteins in ACHN cells treated with Bre 10 µM or 20 µM for 48 h. (C) qPCR analysis of ISGs in ACHN treated with Bre 10 µM compared to non-treated control cells (NT). Data are shown as the means ± SD ( n =3). (D) Representative micrographs of immunofluorescence staining for mitoSOX in indicated cells with or without the addition of each dNs 10 µM and EHNA 5 µM for 48 h. The fluorescence intensity was calculated by ImageJ ( n =3). (E) Cellular growth of ACHN control (CTL) and GLDC knock-downed cells (shGLDC) after the addition of each dNs 10 µM and EHNA 5 µM to inhibit of degradation of dATP in cell culture. Data are shown as the means ± SD ( n =3). (F) Western blot of indicated proteins in ACHN CTL and shGLDC cells 48 h after the addition of each dNs 10 µM and EHNA 5 µM. p values were calculated using two-tailed unpaired Student t -test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Brequinar (Bre) sodium was purchased from Tocris, Bristol, UK.

Techniques: Inhibition, CCK-8 Assay, Western Blot, Control, Immunofluorescence, Staining, Fluorescence, Cell Culture, Two Tailed Test

Journal: Cancer cell

Article Title: Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma.

doi: 10.1016/j.ccell.2022.10.009

Figure Lengend Snippet: Figure 2. Attenuation of key metabolic pathways selectively targets BTICs in MYC-amplified group 3 medulloblastoma (A) Immunoblot confirmation of DHODH and PGD KO in SU_MB002. Cropped from full blots in Figure S6. (B) Assessment of tumor cell growth kinetics measured by time course PrestoBlue reduction cell viability assays in DHODH, PGD (2 sgRNAs tested per gene), and AAVS1 KO SU_MB002 tumor cells. Comparisons of cell viability after 120 h were made via one-way ANOVA and post hoc Tukey’s test, ****p < 0.0001, n = 4 independent replicates. (C) Representative phase-contrast microscopy images of SU_MB002 cells harboring AAVS1, PGD, and DHODH KO. Scale: 200 mm (D) Quantification of tumor spheres formed by DHODH-KO A, PGD-KO A, and AAVS1 KO SU_MB002 tumor cells. One-way ANOVA and post hoc Tukey’s test, ****p < 0.0001, n = 4 independent replicates. (E) Assessment of tumor cell growth kinetics measured by time course PrestoBlue reduction cell viability assays in DHODH-KO A, PGD-KO A, and AAVS1 KO HD- MB03 tumor cells. (F) Quantification of tumor spheres formed by DHODH-KO A, PGD-KO A, and AAVS1 KO HD-MB03 tumor cells. (G) Immunoblot confirmation of KO of DHODH-KO A and PGD-KO A in NSC197 cells. Cropped from full blots in Figure S6. (H) Assessment of cell viability of DHODH-KO A, PGD-KO A, and AAVS1 KO NSC197 cells expressed as percentage of residual PrestoBlue reduction (percentage of AAVS1). (I) Assessment of cell viability of SU_MB002 tumor cells treated with PGD inhibitor S3 (20 mM) or its vehicle (0.5% DMSO). (J and K) Dose-response curves of cell viability after a 72-h treatment with BAY2402234 (J) and Brequinar (BQR; K). Cell viability (residual PrestoBlue reduction) normalized to vehicle-treated cells. IC50 calculations made via nonlinear regression. (L) Kaplan-Meier survival analysis of mice xenografted with DHODH and AAVS1 KO tumor cells (HD-MB03 and SU_MB002). Log-rank tests, **p = 0.0053, n = 5–6 mice. (M) Representative H&E-stained sections of brains isolated from time-matched specimens. Scale: 7mm. (N) Tumor area (mm2) of time-matched brain tissue sections. Unpaired t test; *p = 0.01; ***p = 0.001, n = 3–4 mice. All error bars represent standard error of the mean calculated across four technical replicates.

Article Snippet: BAY2402234 was purchased from Cayman Chemical Suppliers (Catalogue #33259), Brequinar sodium was purchased from Tocris (Catalogue #6196), and PTC299 was procured via a scientific research agreement with PTC therapeutics.

Techniques: Western Blot, Microscopy, Staining, Isolation