Journal: Journal of Thoracic Disease

Article Title: 4-phenylbutyric acid re-trafficking hERG/G572R channel protein by modulating the endoplasmic reticulum stress-associated chaperones and endoplasmic reticulum-associated degradation gene

doi: 10.21037/jtd-23-1252

Figure Lengend Snippet: 4-PBA increased the current density of the hERG/G572R channel. (A) Representative whole-cell patch-clamp current traces were recorded using the top voltage protocol with and without 5 mM 4-PBA treatment. (B) Current density-voltage relationship of tail current density (pA/pF) in WT/G572R and WT/G572R + 4-PBA drug treatment groups. (C) Statistical graph of tail current at 0, 10, and 20 mV. Each scatter represents independent cell data. WT/G572R, n=5; WT/G572R + 4-PBA, n=5. **, P<0.01; ***, P<0.001; ****, P<0.0001. WT, wild type; 4-PBA, 4-phenylbutyric acid; hERG, human ether-à-go-go-related gene.

Article Snippet: 4-PBA (cat. no. P831259) was purchased from Macklin (Shanghai, China).

Techniques: Patch Clamp

Journal: Journal of Thoracic Disease

Article Title: 4-phenylbutyric acid re-trafficking hERG/G572R channel protein by modulating the endoplasmic reticulum stress-associated chaperones and endoplasmic reticulum-associated degradation gene

doi: 10.21037/jtd-23-1252

Figure Lengend Snippet: 4-PBA upregulated the expression of the hERG/G572R channel. (A) WB results showing protein expression of WT, G572R, and WT/G572R transfected cells, and protein expression of G572R and WT/G572R transfected cells after 4-PBA treatment. (B) Optical density analysis of the mature (155 kDa) form of hERG protein in 4-PBA-untreated control and 4-PBA-treated experimental groups. (C) hERG qPCR mRNA expression in 4-PBA-untreated control and 4-PBA-treated experimental groups. ns, non-significant; *, P<0.05; **, P<0.01; ****, P<0.0001. hERG, human ether-à-go-go-related gene; 4-PBA, 4-phenylbutyric acid; WT, wild type; WB, western blot; qPCR, quantitative real-time polymerase chain reaction; mRNA, messenger RNA.

Article Snippet: 4-PBA (cat. no. P831259) was purchased from Macklin (Shanghai, China).

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of Thoracic Disease

Article Title: 4-phenylbutyric acid re-trafficking hERG/G572R channel protein by modulating the endoplasmic reticulum stress-associated chaperones and endoplasmic reticulum-associated degradation gene

doi: 10.21037/jtd-23-1252

Figure Lengend Snippet: 4-PBA inhibited the expression of ATF6, GRP78, GRP94, and CRT/CNX. (A) WB results showing protein expression of ATF6, GRP78, and GRP94 in WT, WT/G572R, and WT/G572R + 5 mM 4-PBA cell lines. (B) Statistical analysis of the WB protein optical density of ATF6 in WT, WT/G572R, WT/G572R + 5 mM 4-PBA. (C) Statistical analysis of the WB protein optical density of GRP78 in WT, WT/G572R, and WT/G572R + 5 mM 4-PBA. (D) Statistical analysis of the WB protein optical density of GRP94 in WT, WT/G572R, and WT/G572R + 5 mM 4-PBA. (E) ATF6 mRNA expression levels in WT, WT/G572R untreated control, and WT/G572R + 4-PBA experimental groups. (F) GRP78 mRNA expression levels in WT, WT/G572R untreated control, and WT/G572R + 4-PBA experimental groups. (G) GRP94 mRNA expression levels in WT, WT/G572R untreated control, and WT/G572R + 4-PBA experimental groups. (H) CRT mRNA expression levels in WT, WT/G572R untreated control, and WT/G572R + 4-PBA experimental groups. (I) CNX mRNA expression levels in WT, WT/G572R untreated control, and WT/G572R + 4-PBA experimental groups. ns, non-significant; *, P<0.05; **, P<0.01; ****, P<0.0001. hERG, human ether-à-go-go-related gene; 4-PBA, 4-phenylbutyric acid; ATF6, activating transcription factor 6; WT, wild type; CRT, calreticulin; CNX, calnexin; WB, western blot; mRNA, messenger RNA.

Article Snippet: 4-PBA (cat. no. P831259) was purchased from Macklin (Shanghai, China).

Techniques: Expressing, Western Blot

Journal: Journal of Thoracic Disease

Article Title: 4-phenylbutyric acid re-trafficking hERG/G572R channel protein by modulating the endoplasmic reticulum stress-associated chaperones and endoplasmic reticulum-associated degradation gene

doi: 10.21037/jtd-23-1252

Figure Lengend Snippet: 4-PBA inhibited ERAD gene HRD1. (A) WB results showing HRD1 protein expression in WT, WT/G572R, and WT/G572R + 5 mM 4-PBA cell lines. (B) Statistical graph of HRD1 protein expression in WT, WT/G572R, and WT/G572R + 5 mM 4-PBA cell lines. (C) Statistical analysis of HRD1 mRNA expression in WT, WT/G572R and WT/G572R + 5 mM 4-PBA cell lines. (D) co-IP of GRP78, GRP94, HRD1, and hERG (WT/G572R)-FLAG channel. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. hERG, human ether-à-go-go-related gene; 4-PBA, 4-phenylbutyric acid; HRD1, 3-hydroxy-3-methylglutaryl coenzyme A reductase degradation protein 1; WT, wild type; WB, western blot; IP, immunoprecipitation; co-IP, co-immunoprecipitation; mRNA, messenger RNA.

Article Snippet: 4-PBA (cat. no. P831259) was purchased from Macklin (Shanghai, China).

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

Journal: JCI Insight

Article Title: Multidimensional analysis and therapeutic development using patient iPSC–derived disease models of Wolfram syndrome

doi: 10.1172/jci.insight.156549

Figure Lengend Snippet: ( A ) A schematic of Wolfram syndrome etiology and the targets to modulate by a combination treatment of 4-PBA and TUDCA (P+T). ( B ) Expression of HiBiT-tagged WFS1 protein after treatment with 500 μM 4-PBA and 50 μM TUDCA (P+T) for 24 hours. NanoLuc levels, expressed from an identical plasmid backbone, were examined ( n = 48, P value by unpaired t test). ( C ) (Left) Representative blotting images of WFS1 and α-Tubulin in iPSCs treated with or without P+T for 48 hours. (Right) Quantification of WFS1 protein levels normalized with α-Tubulin. ( n = 3, * P < 0.05 by unpaired t test compared with Ctrl.) ( D ) Relative mRNA levels of WFS1 in iPSCs treated with or without P+T for 48 hours ( n = 5, ** P < 0.01 by unpaired t test compared with Ctrl). ( E ) Representative immunofluorescence images of neural progenitor cell (NPC) markers in NPCs differentiated from patient-derived iPSCs. Scale bar: 100 μm. ( F ) Quantitative PCR analysis of ER stress–related genes in NPCs treated with or without P+T for 48 hours. ( n = 6, * P < 0.05, ** P < 0.01, and **** P < 0.0001 by unpaired t test compared with Ctrl.) ( G ) Mitochondrial respiration of NPCs treated with or without P+T for 48 hours represented as percentage of baseline oxygen consumption rate (OCR) measurements. Respiration was interrogated by measuring changes in relative OCR multiple times, every 8.5 minutes, after injection with oligomycin (OM), FCCP, and antimycin A (AA)/rotenone (R) ( n = 3, W024: *** P < 0.001, W392: * P < 0.05, and W121: * P < 0.05 by unpaired t test compared with Ctrl AUC). ( H ) Caspase-3/7 activity normalized by cell viability in NPCs treated with or without either of 4-PBA, TUDCA, or P+T for 48 hours. ( n = 7; *** P < 0.001 and **** P < 0.0001 by 1-way ANOVA compared with Ctrl; # P < 0.05, ## P < 0.01, and #### P < 0.0001 by 1-way ANOVA.)

Article Snippet: We obtained 4-PBA and TUDCA from Amylyx Pharmaceuticals Inc. We dissolved 4-PBA and TUDCA in PBS and used them at final concentrations of 500 μM and 50 μM, respectively.

Techniques: Expressing, Plasmid Preparation, Immunofluorescence, Derivative Assay, Real-time Polymerase Chain Reaction, Injection, Activity Assay

Journal: JCI Insight

Article Title: Multidimensional analysis and therapeutic development using patient iPSC–derived disease models of Wolfram syndrome

doi: 10.1172/jci.insight.156549

Figure Lengend Snippet: ( A ) Intraperitoneal glucose tolerance test (IP-GTT) with WT or Wfs1 -KO mice at baseline and 1 month after feeding with either control chow or food containing 4-PBA: 0.338% and TUDCA: 0.225% (P+T chow). ( B ) AUCs of the IP-GTT (KO, Ctrl: n = 12; KO, P+T: n = 12; WT: n = 7; ** P < 0.01 and *** P < 0.001 by 1-way ANOVA; ## P < 0.01 and #### P < 0.0001 by 1-way ANOVA compared with WT: Baseline; †††† P < 0.0001 by 1-way ANOVA compared with WT: 1 month).

Article Snippet: We obtained 4-PBA and TUDCA from Amylyx Pharmaceuticals Inc. We dissolved 4-PBA and TUDCA in PBS and used them at final concentrations of 500 μM and 50 μM, respectively.

Techniques: