Journal: Environmental Health Perspectives

Article Title: Effects of Inorganic Arsenic on Human Prostate Stem-Progenitor Cell Transformation, Autophagic Flux Blockade, and NRF2 Pathway Activation

doi: 10.1289/EHP6471

Figure Lengend Snippet: The role of NRF2 plays in iAs-induced transformation. (A) GSEA data of NRF2 pathway genes comparing prostate organoids (PO) and spheres (PS) derived from primary human prostate epithelial cells (PrEC) and treated with vehicle (PO-V vs. PS-V) or 1 μ M arsenic (PO-As vs. PS-As). (B) Expression of prostate epithelial differentiation genes in 2-dimensional culture PrEC (PrEC-2D) with NRF2 knockdown (shNRF2). Data shown are mean ± SEM ( n = 3 ); * p < 0.05 vs. shRNA control (shLuc). (C) Sphere formation capability of PrEC with NRF2-knockdown, D7PS: day-7 spheres. Data shown are mean ± SEM ( n = 4 ); * p < 0.05 vs. vehicle. (D) Quantification of sphere formation capability of PrEC treated with NRF2-inducer Oltipraz ( 10 μ M ) for 7 d. D7PS: day-7 prostate spheres. Data shown are mean ± SEM ( n = 4 ); * p < 0.05 vs. vehicle. (E) mRNA level of NRF2 gene and NRF2 pathway marker genes. Tet-On NRF2 expression was induced in PrEC spheres by lentivirus. Spheres were culture − / + doxycycline (Dox, 0.5 ug / mL ) for 7 d (Tet-ON-NRF2 PrEC-D7PS) to induce NRF2 overexpression. Data shown are mean ± SEM ( n = 4 ); * p < 0.05 vs. vehicle. (F) Quantification of sphere formation capability of PrEC with Tet-On overexpression of NRF2. Data shown are mean ± SEM ( n = 4 ); * p < 0.05 vs. vehicle. (G) Soft-agar colony formation assay of RWPE1 with indicated treatment. Note: iAs, inorganic arsenic; shLuc, RWPE1 with shRNA targeting luciferase gene, negative control; shNRF2, RWPE1 with NRF2 knockdown. Data shown are mean ± SEM ( n = 4 ); * p < 0.05 vs. shRNA control (shLuc).

Article Snippet: This was subcloned to a Tet-On overexpression lentiviral vector pLIX_402 (a gift from David Root, Addgene plasmid 41393) with a C-terminal HA tag. mCherry-EGFP-LC3B CDS were subcloned from pBabe-puro mCherry-EGFP-LC3B (a gift from Jayanta Debnath, Addgene plasmid 22418) to pLVX-puro (Clontech; Cat. No. 632164).

Techniques: Transformation Assay, Derivative Assay, Expressing, Knockdown, shRNA, Control, Marker, Over Expression, Soft Agar Assay, Luciferase, Negative Control

Journal: Environmental Health Perspectives

Article Title: Effects of Inorganic Arsenic on Human Prostate Stem-Progenitor Cell Transformation, Autophagic Flux Blockade, and NRF2 Pathway Activation

doi: 10.1289/EHP6471

Figure Lengend Snippet: Arsenic effects on lysosome acidification and expression of V-ATPase subunit VMA5. (A) LysoHunt staining showing acidification of organelles from day-4 spheres of primary human prostate epithelial cells (PrEC-D4PS) cultured on low-attached plate with indicated treatment. Higher intensity of red signal from LysoHunt indicates higher acidification. BafA1, bafilomycin A1, V-ATPase inhibitor, 100 nM treatment for 2 h; iAs, 1 μ M iAs treatment for 4 d; Veh, vehicle. Quantification data are mean ± SEM ( n = 10 – 13 spheres from wells of same low-attach plate); * p < 0.05 vs. vehicle. (B) mRNA level of VMA5 gene in D7PS of RWPE1 and PrEC treated with 1 μ M iAs for 7 d. Mean ± SEM ( n = 4 ); * p < 0.05 vs. vehicle. (C) Immunoblot of VMA5 protein in PrEC-D7PS treated with 1 μ M iAs for 7 d. Mean ± SEM ( n = 3 ); * p < 0.05 vs. vehicle. (D) mRNA level of VMA5 in PrEC with shRNA targeting VMA5 (shVMA5). Mean ± SEM ( n = 4 ); * p < 0.05 vs. shRNA control (shLuc). (E) LysoHunt staining showing acidification of organelles from day-4 spheres of primary human prostate epithelial cells (PrEC-D4PS) with VMA5 gene knockdown (shVMA5). Quantitative data are mean ± SEM ( n = 10 – 15 ); * p < 0.05 vs. shRNA control (shLuc). (F) Immunoblotting of p62 and LC3B from PrEC-D7PS with VMA5 gene knockdown (shVMA5). Mean ± SEM ( n = 4 ); * p < 0.05 vs. shRNA control (shLuc). (G) Immunoblot of indicated protein in PrEC-D7PS with Tet-On VMA5 overexpression treated with 1 μ M iAs for 7 d. Doxycycline (Dox, 0.5 ug / mL ) was added to induce VMA5 overexpression for 7 d. Quantification data are mean ± SEM ( n = 3 ); * p < 0.05 vs. vehicle control.

Article Snippet: This was subcloned to a Tet-On overexpression lentiviral vector pLIX_402 (a gift from David Root, Addgene plasmid 41393) with a C-terminal HA tag. mCherry-EGFP-LC3B CDS were subcloned from pBabe-puro mCherry-EGFP-LC3B (a gift from Jayanta Debnath, Addgene plasmid 22418) to pLVX-puro (Clontech; Cat. No. 632164).

Techniques: Expressing, Staining, Cell Culture, Western Blot, shRNA, Control, Knockdown, Over Expression