Journal: The FASEB Journal

Article Title: Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

doi: 10.1096/fj.201900893r

Figure Lengend Snippet: FIGURE 5 Albumin induces ER stress in vitro and in vivo. Relative mRNA and protein levels of ER stress response genes as measured by qPCR (N = 10), (A) and western blot (N = 8), (B), respectively, in HEK-293 overexpressing cells exposed to 10 mg/mL BSA for 24 hours. Relative mRNA and protein levels of ER stress response genes as measured by qPCR (N = 10), (C) and western blot (N = 8), (D), respectively, in the HK-2 cells exposed to 10 mg/mL BSA for 24 hours. Relative mRNA levels of ER stress response genes in the cortex of control (N = 13) and proteinuric mice (N = 16) 7 days post-injection (E). Representative immunohistochemical staining of ATF4 and ATF3 transcription factors in kidney sections (F). The mRNA levels are normalized to RPLP0 gene expression and the protein levels to β-actin. Mean ± SD, multiple t test without correction *P < .05

Article Snippet: HK-2 or FNL-7 cells were transiently transfected with plasmids expressing the human ATF4 (pRK-FLAG-ATF4, #26114; Addgene, Cambridge, MA) and human ATF3 (pRK-FLAG-ATF3, #26115; Addgene) transcription factors with the use of FuGENE HD as described before.

Techniques: In Vitro, In Vivo, Western Blot, Control, Injection, Immunohistochemical staining, Staining, Gene Expression

Journal: The FASEB Journal

Article Title: Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

doi: 10.1096/fj.201900893r

Figure Lengend Snippet: FIGURE 6 ATF4 and ATF3 regulate Klotho expression. Relative mRNA and protein levels of Klotho, ATF4 and ATF3 as measured by qPCR and western blot, respectively, in HK-2 cells transiently overexpressing the human ATF4 transcription factor (two experiments), (A) or the human ATF3 transcription factor (three experiments), (B). Relative luminescence in the FNL-7 cell line expressing the human Klotho promoter driving firefly luciferase expression in control and cells overexpressing human ATF4, ATF3, or both factors (N = 6), (C). The HK-2 cells were also transduced with shRNA lentiviral vectors targeting ATF4 or ATF3 and the relative mRNA levels of Klotho as well as the protein levels were assessed by qPCR (N = 8/8/4), (D) or western blot analysis (N = 3), (E), respectively, in the presence or absence of albumin in comparison to the scrambled shRNA vector. The silencing efficiency was verified by western blot analysis (N = 4/3), (F). The mRNA levels are normalized to RPLP0 gene expression and the protein levels to β-actin or Ponceau staining. Mean ± SD, unpaired two-tailed t test or multiple t test without correction or one-way ANOVA *P < .05

Article Snippet: HK-2 or FNL-7 cells were transiently transfected with plasmids expressing the human ATF4 (pRK-FLAG-ATF4, #26114; Addgene, Cambridge, MA) and human ATF3 (pRK-FLAG-ATF3, #26115; Addgene) transcription factors with the use of FuGENE HD as described before.

Techniques: Expressing, Western Blot, Luciferase, Control, Transduction, shRNA, Comparison, Plasmid Preparation, Gene Expression, Staining, Two Tailed Test