Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 6. NRF2 regulates the stability of the TAp73 protein. (A and B) TAp73 and G6PD mRNA levels in control and NRF2-knockdown PANC-1 (A) and U2OS (B) cells cultured under matrix-detached conditions for the indicated durations or treated with the indicated doses of H2O2. (C to H) U2OS cells (C, D, G, and H) or TAp73-overexpressing U2OS cells (E and F) were treated with control or NRF2 siRNA (C to H) in the presence of CHX alone (C to F) or CHX plus MG132 (G and H) as indicated. Shown are representative immunoblots (C, E, and G) and quantification of relative TAp73/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio in each sample, with the half-life of TAp73 indicated by dashed vertical lines (D, F, and H). (I) Human embryonic kidney (HEK) 293T cells were transfected with control siRNA, NRF2, or PIRH2 siRNA, along with hemagglutinin-ubiquitin (HA-Ub) and/or Flag-TAp73 expression vectors as indicated. Cells were treated with MG132 for 24 hours. After denaturing immunoprecipitation (d-IP) with the anti-Flag antibody, immunoprecipitates and whole-cell lysates were analyzed by Western blot. (J to M) Control, NRF2-overexpressing (J and K), and NRF1-overexpressing (L and M) U2OS cells were treated with CHX for the indicated times and assayed for protein expression (J and L) with quantification of relative TAp73/G6PD ratios shown in (K) and (M). Data are means ± SD [n = 4 for (A) and (B) and 3 for the rest] and are representative of three independent experiments. **P < 0.01 and ***P < 0.001; unpaired Student’s t test.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Control, Knockdown, Cell Culture, Western Blot, Transfection, Ubiquitin Proteomics, Expressing, Immunoprecipitation

Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 7. NRF2 suppresses the expression of the PIRH2 gene. (A to D) Protein (A and B) and mRNA (C and D) levels of TAp73 regulators in control and NRF2-knockdown PANC-1 or U2OS cells. (E to H) PIRH2 and TAp73 protein levels (E and F) and PIRH2 mRNA levels (G and H) in PANC-1 (E and G) and U2OS (F and H) cells with PIRH2 or NRF2 knockdown (by siRNA or shRNA) (E and F) or overexpression (E to H). (I) Flag-PIRH2 was transfected into control or NRF2-overexpressing HEK293T cells. Cells were assayed for protein expression. (J) HA-TAp73 was expressed in control or NRF2-overexpressing HEK293T cells. Interaction between HA-TAp73 and endogenous PIRH2 was detected by coimmunoprecipitation with anti-HA antibody. WCL, whole cell lysates. (K to P) Control and PIRH2-knockdown PANC-1 cells were cultured under matrix-detached conditions for the indicated times (K and M to P) or for 12 hours (L). Cells were assayed for protein expression (K), isotopic incorporation (L), NADP+ and NADPH levels (M), the NADPH/NADP+ ratio (N), ROS content (O), and viability (P). The isotopic tracing experiment in (L) was done together with that in Fig. 4C. (Q) Schematic diagram of the promoter region of PIRH2 (PR-PIRH2) as well as wild-type (PIRH2) and mutated (m-PIRH2) PIRH2 promoter sequences that were cloned into pGL3 vector. (R) NRF2-binding consensus sequence, sequence of putative ARE3, and its corresponding mutation. (S) ChIP assay of NRF2 binding to putative AREs in HEK293T cells. The enrichment fold was normalized with the corresponding immunoglobulin G (IgG) controls. (T) NRF2-mediated suppression of luciferase reporter gene driven by PIRH2 but not m-PIRH2 in HEK293T cells. Data are means ± SD [n = 4 for (C), (D), (G), and (H) and 3 for the rest] and are representative of two (R) and three (the rest) independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA for (L) and (M) and unpaired Student’s t test for the rest.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Expressing, Control, Knockdown, shRNA, Over Expression, Transfection, Cell Culture, Clone Assay, Plasmid Preparation, Binding Assay, Sequencing, Mutagenesis, Luciferase

Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 8. Redox stress stimulates TAp73 expression via the CHK1-E2F1 pathway, and both CHK1-E2F1 and NRF2-PIRH2 pathways regulate TAp73 and G6PD to promote anchorage-independent growth. (A to H) PANC-1 cells treated with control, E2F1, and/or NRF2 siRNAs were cultured under indicated conditions. Cells were assayed for protein expression (A and H), mRNA levels (B), isotope incorporation (C), NADP+ and NADPH levels (D), the NADPH/NADP+ ratio (E), ROS content (F), and via- bility (G). The isotopic tracing experiments in (C) were done together with that in Fig. 4C. (I) Schematic diagram shows signaling network that regulates G6PD for redox homeostasis. (J to L) PANC-1 cells with knockdown of the individual gene were assayed for protein expression (J), adherent growth (K), and soft-agar colony formation (L) (related to fig. S7C). (M to O) Control and G6PD-overexpressing MIA PaCa-2 cells with or without NADK1 knockdown were assayed for protein expression (M), adherent growth (N), and soft-agar colony formation (O) (related to fig. S7G). (P and Q) PANC-1 cells with or without NADK1 knockdown were assayed for protein expression (P) and soft-agar colony formation (Q) (related to fig. S7M). (R) Soft-agar colony formation by control, G6PD-overexpressing, and G6PDK171Q-overexpressing MIA PaCa-2 cells. Protein expression is shown in Fig. 2P. (S) Levels of G6PD and its regulators in a panel of pancreatic cancer cells. (T to V) MIA PaCa-2 and PANC-1 cells were cultured under matrix-detached conditions for the indicated durations and assayed for protein expression (T), ROS content (U), and viability (V). Data are means ± SD [n = 4 for (B) and 3 for the rest] and are representative of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA for (C), (D), (L), (O), and (R) and unpaired Student’s t test for the rest.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Expressing, Control, Cell Culture, Knockdown

Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 10. Expression of G6PD and its regulators in primary and metastatic pancreatic cancers. (A to F) TMAs containing primary and metastatic pancreatic tumors were analyzed by IHC for expression of G6PD (A), NRF2 (B), E2F1 (C), p73 (D), PIRH2 (E), and NADK1 (F). The representative image of IHC staining (left) and intensity of antibody staining (a.u.) (right) are shown. Scale bars, 60 m. (G to J) Correlation coefficient analysis for the expression of NRF2 (a.u., same below) and PIRH2 (n = 45) (G), PIRH2 and p73 (n = 45) (H), E2F1 and p73 (n = 43) (I), and p73 and G6PD (n = 46) (J). The r and P values of Pearson correlation coefficient are shown. Data are means ± SD (n as indicated). *P < 0.05, **P < 0.01, and ***P < 0.001; unpaired Student’s t test.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 5. The TAp73-G6PD axis is a major downstream effector of NRF2. (A to G) PANC-1 (A to F) or U2OS (G) cells treated with control or NRF2 siRNA were cultured under matrix-attached or matrix-detached conditions as indicated or for 12 hours (A to F) or exposed to different doses of H2O2 for 48 hours (G). Cells were assayed for protein expression (A and G), isotopic incorporation (B), NADP+ and NADPH levels (C), the NADPH/NADP+ ratio (D), ROS content (E), and viability (F). The isotopic tracing experiment in (B) was done together with that in Fig. 4C. (H to L) PANC-1 cells harboring control or G6PD expression vectors were treated with control or NRF2 siRNA and cultured under indicated conditions. Cells were assayed for protein expression (H), NADP+ and NADPH levels (I), the NADPH/NADP+ ratio (J), ROS content (K), and viability (L). (M to P) Protein expression (M and O) and mRNA levels of NRF2 target genes involved in antioxidant defense (N and P) in MIA PaCa-2 cells harboring control or G6PD expression vectors (M and N) or PANC-1 cells treated with control or G6PD siRNA (O and P) that were cultured under indicated conditions. Data are means ± SD [n = 4 for (N) and (P) and 3 for the rest] and are representative of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA for (B), (C), and (I) and unpaired Student’s t test for the rest. GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; GPx2, glutathione peroxidase 2; NQO1, NAD(P)H quinone dehydrogenase 1; PRX1, Peroxiredoxin 1; FTL, ferritin light chain; TRX, thioredoxin.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Control, Cell Culture, Expressing

Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 6. NRF2 regulates the stability of the TAp73 protein. (A and B) TAp73 and G6PD mRNA levels in control and NRF2-knockdown PANC-1 (A) and U2OS (B) cells cultured under matrix-detached conditions for the indicated durations or treated with the indicated doses of H2O2. (C to H) U2OS cells (C, D, G, and H) or TAp73-overexpressing U2OS cells (E and F) were treated with control or NRF2 siRNA (C to H) in the presence of CHX alone (C to F) or CHX plus MG132 (G and H) as indicated. Shown are representative immunoblots (C, E, and G) and quantification of relative TAp73/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio in each sample, with the half-life of TAp73 indicated by dashed vertical lines (D, F, and H). (I) Human embryonic kidney (HEK) 293T cells were transfected with control siRNA, NRF2, or PIRH2 siRNA, along with hemagglutinin-ubiquitin (HA-Ub) and/or Flag-TAp73 expression vectors as indicated. Cells were treated with MG132 for 24 hours. After denaturing immunoprecipitation (d-IP) with the anti-Flag antibody, immunoprecipitates and whole-cell lysates were analyzed by Western blot. (J to M) Control, NRF2-overexpressing (J and K), and NRF1-overexpressing (L and M) U2OS cells were treated with CHX for the indicated times and assayed for protein expression (J and L) with quantification of relative TAp73/G6PD ratios shown in (K) and (M). Data are means ± SD [n = 4 for (A) and (B) and 3 for the rest] and are representative of three independent experiments. **P < 0.01 and ***P < 0.001; unpaired Student’s t test.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Control, Knockdown, Cell Culture, Western Blot, Transfection, Ubiquitin Proteomics, Expressing, Immunoprecipitation

Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 7. NRF2 suppresses the expression of the PIRH2 gene. (A to D) Protein (A and B) and mRNA (C and D) levels of TAp73 regulators in control and NRF2-knockdown PANC-1 or U2OS cells. (E to H) PIRH2 and TAp73 protein levels (E and F) and PIRH2 mRNA levels (G and H) in PANC-1 (E and G) and U2OS (F and H) cells with PIRH2 or NRF2 knockdown (by siRNA or shRNA) (E and F) or overexpression (E to H). (I) Flag-PIRH2 was transfected into control or NRF2-overexpressing HEK293T cells. Cells were assayed for protein expression. (J) HA-TAp73 was expressed in control or NRF2-overexpressing HEK293T cells. Interaction between HA-TAp73 and endogenous PIRH2 was detected by coimmunoprecipitation with anti-HA antibody. WCL, whole cell lysates. (K to P) Control and PIRH2-knockdown PANC-1 cells were cultured under matrix-detached conditions for the indicated times (K and M to P) or for 12 hours (L). Cells were assayed for protein expression (K), isotopic incorporation (L), NADP+ and NADPH levels (M), the NADPH/NADP+ ratio (N), ROS content (O), and viability (P). The isotopic tracing experiment in (L) was done together with that in Fig. 4C. (Q) Schematic diagram of the promoter region of PIRH2 (PR-PIRH2) as well as wild-type (PIRH2) and mutated (m-PIRH2) PIRH2 promoter sequences that were cloned into pGL3 vector. (R) NRF2-binding consensus sequence, sequence of putative ARE3, and its corresponding mutation. (S) ChIP assay of NRF2 binding to putative AREs in HEK293T cells. The enrichment fold was normalized with the corresponding immunoglobulin G (IgG) controls. (T) NRF2-mediated suppression of luciferase reporter gene driven by PIRH2 but not m-PIRH2 in HEK293T cells. Data are means ± SD [n = 4 for (C), (D), (G), and (H) and 3 for the rest] and are representative of two (R) and three (the rest) independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA for (L) and (M) and unpaired Student’s t test for the rest.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Expressing, Control, Knockdown, shRNA, Over Expression, Transfection, Cell Culture, Clone Assay, Plasmid Preparation, Binding Assay, Sequencing, Mutagenesis, Luciferase

Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 8. Redox stress stimulates TAp73 expression via the CHK1-E2F1 pathway, and both CHK1-E2F1 and NRF2-PIRH2 pathways regulate TAp73 and G6PD to promote anchorage-independent growth. (A to H) PANC-1 cells treated with control, E2F1, and/or NRF2 siRNAs were cultured under indicated conditions. Cells were assayed for protein expression (A and H), mRNA levels (B), isotope incorporation (C), NADP+ and NADPH levels (D), the NADPH/NADP+ ratio (E), ROS content (F), and via- bility (G). The isotopic tracing experiments in (C) were done together with that in Fig. 4C. (I) Schematic diagram shows signaling network that regulates G6PD for redox homeostasis. (J to L) PANC-1 cells with knockdown of the individual gene were assayed for protein expression (J), adherent growth (K), and soft-agar colony formation (L) (related to fig. S7C). (M to O) Control and G6PD-overexpressing MIA PaCa-2 cells with or without NADK1 knockdown were assayed for protein expression (M), adherent growth (N), and soft-agar colony formation (O) (related to fig. S7G). (P and Q) PANC-1 cells with or without NADK1 knockdown were assayed for protein expression (P) and soft-agar colony formation (Q) (related to fig. S7M). (R) Soft-agar colony formation by control, G6PD-overexpressing, and G6PDK171Q-overexpressing MIA PaCa-2 cells. Protein expression is shown in Fig. 2P. (S) Levels of G6PD and its regulators in a panel of pancreatic cancer cells. (T to V) MIA PaCa-2 and PANC-1 cells were cultured under matrix-detached conditions for the indicated durations and assayed for protein expression (T), ROS content (U), and viability (V). Data are means ± SD [n = 4 for (B) and 3 for the rest] and are representative of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001; two-way ANOVA for (C), (D), (L), (O), and (R) and unpaired Student’s t test for the rest.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Expressing, Control, Cell Culture, Knockdown

Journal: Science advances

Article Title: G6PD-mediated increase in de novo NADP + biosynthesis promotes antioxidant defense and tumor metastasis.

doi: 10.1126/sciadv.abo0404

Figure Lengend Snippet: Fig. 10. Expression of G6PD and its regulators in primary and metastatic pancreatic cancers. (A to F) TMAs containing primary and metastatic pancreatic tumors were analyzed by IHC for expression of G6PD (A), NRF2 (B), E2F1 (C), p73 (D), PIRH2 (E), and NADK1 (F). The representative image of IHC staining (left) and intensity of antibody staining (a.u.) (right) are shown. Scale bars, 60 m. (G to J) Correlation coefficient analysis for the expression of NRF2 (a.u., same below) and PIRH2 (n = 45) (G), PIRH2 and p73 (n = 45) (H), E2F1 and p73 (n = 43) (I), and p73 and G6PD (n = 46) (J). The r and P values of Pearson correlation coefficient are shown. Data are means ± SD (n as indicated). *P < 0.05, **P < 0.01, and ***P < 0.001; unpaired Student’s t test.

Article Snippet: 8, eabo0404 (2022) 20 July 2022 19 of 24 12-myristate 13-acetate, and gallotannin (Cayman Chemical, Ann Arbor, MI); protein A/G agarose beads (Santa Cruz Biotechnology); low-temperature melting agarose and MEGM BulletKit Growth Media (Lonza, Basel, Switzerland); folic acid (MP Biomedicals, Solon, OH); Dulbecco’s modified Eagle’s medium (DMEM) Base (without l-glutamine, l-cystine, glucose, phenol red, and sodium pyruvate) (United States Biological, Salem, MA); 4,6-diamidino- 2- phenylindole (Vector Laboratories, Burlingame, CA); Dako Dual Endogenous Enzyme Block (DAKO, Little Ferry, NJ); and Novocastra Epitope Retrieval Solutions (Leica Biosystems, Buffalo Grove, IL). siRNAs for NRF2, PIRH2, p73, G6PD, and IDH1 were purchased from Thermo Fisher Scientific; siRNA for NADK1, E2F1, and ALDH1L2 was purchased from Santa Cruz Biotechnology; and siRNA for NADK2 was purchased from Horizon Discovery (St. Louis, MO).

Techniques: Expressing, Immunohistochemistry, Staining