fret-based biosensor plasmids  (Promega)

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 1 Expression of the ER- resident GPx8 attenuates palmitate (PA)- mediated perturbation of intraorganellar Ca2+ dynamics. (A) Average trace of time-dependent changes in cytosolic free Ca2+ in control and GPx8 expressing INS-1E cells perifused under basal conditions (KR + 3 mmol/L glucose + 1% FFA-free BSA), followed by 0.5 mmol/L PA-containing KR solution for 5 min. Bars on the right represent the average peak amplitude of cytosolic free Ca2+ in the presence of PA. (B) Average curve of ER luminal and (C) mitochondrial Ca2+ dynamics in control and GPx8 cells transfected with the site-specific FRET-based Ca2+ sensors D1ER or 4mtD3cpv before and after perifusion with 0.5 mmol/L PA. The respective bars show the average depletion of ER luminal Ca2+ and the average peak amplitude of mitochondrial Ca2+ levels in the presence of PA, respectively. Data are means ± SEM of 7–10 independent experiments; *p < .05, **p < .01, ***p < .001 compared with INS-1E control cells (Student's t-test, unpaired, two-tailed).

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Expressing, Control, Transfection, Two Tailed Test

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 2 Oxidative metabolism of palmitate (PA) is required for the disruption of ER Ca2+ homeostasis. (A) Average trace of ER Ca2+ dynamics upon continuous perifusion with 0.5 mmol/L 2-BromoPA, followed by 0.5 mmol/L PA in INS-1E cells, and (B) after co-stimulation with 0.5 mmol/L 2-BromoPA +0.5 mmol/L PA. Data are means ± SEM of 3 independent experiments.

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Disruption

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 3 Expression of the ER-resident GPx8 has no impact on carbachol (CCH) or thapsigargin (TG)-evoked ER Ca2+ depletion. (A and C) Average trace of time-dependent changes in cytosolic free-Ca2+ in control and GPx8 expressing INS-1E cells perifused for 5 min under basal conditions (3 mmol/L glucose-containing KR), followed by 5 min perifusion with 0.25 μmol/L TG or 0.1 mmol/L CCH. Bars on the right represent the average peak amplitude of cytosolic free-Ca2+ in the presence of TG or CCH. (B and D) show the average curves of ER luminal free-Ca2+ under the same perifusion conditions. The respective bars show the average depletion of ER luminal Ca2+ in the presence of TG or CCH. Data are means ± SEM of 5–6 independent experiments.

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Expressing, Control

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 4 Expression of the ER-resident GPx8 diminishes palmitate (PA)-mediated reduction of the Ca2+-ATPase activity. (A) Effects of PA pre-exposure on cytosolic free-Ca2+ in control and GPx8 expressing INS-1E cells. Control and GPx8 expressing INS-1E cells were pre-incubated with 0.5 mmol/L PA for 24 h, loaded with the ratiometric Ca2+ indicator Fura-2/AM, and then subjected to perifusion with 0.25 μmol/L TG for 5 min. Each point on the curve represents means ± SEM of 4–6 independent experiments. (B) INS-1E control and GPx8 expressing cells were incubated under control conditions or with PA (0.5 and 1.0 mmol/L). After 24 h, RNA was isolated and relative gene expression of SERCA2b was analyzed by RT-qPCR and normalized to the housekeeping genes ß-actin, peptidylprolyl isomerase A, and ribosomal protein L32. Data are means ± SEM of 4 independent experiments. (C) Representative trace of ER luminal Ca2+ upon perifusion under basal conditions, 1 μmol/L ionomycin for 5 min, 100 μmol/L EGTA for 3 min, followed by 5 min perifusion with 1 mmol/L Ca2+- containing KR in INS-1E control- and GPx8 expression before and after pre-incubation with PA for 4 and 24 h. (D) Half-time (t1/2) of ER Ca2+ reuptake in INS-1E control- and GPx8 expressing cells treated under basal conditions or with PA for 4 or 24 h. Data are means ± SEM of 4 independent experiments; *p < .05, compared with INS-1E control cells (ANOVA/Dunnett's-test).

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Expressing, Activity Assay, Control, Incubation, Isolation, Gene Expression, Quantitative RT-PCR

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 5 An intact GPx8 catalytic activity is required for protection against palmitate (PA)-induced ER Ca2+ dysregulation and toxicity. (A) Scheme depicting the Site-Directed Mutagenesis of GPx8 cysteine 79 by serine and cysteine 108 by alanine, respectively. (B) Immunoblot showing the expression of GPx8 mutant variants in stably transfected INS-1E cells compared to control cells. (C) Control INS-1E, GPx8-WT, GPx8C79S, and GPx8C79S/C108A transfected INS-1E cells were incubated under control conditions or with increasing PA concentrations for 24 h. Thereafter, viability was determined by MTT assay. Data are means ± SEM of 4–6 independent experiments; *p < .05, **p < .01, compared with control cells incubated under the same conditions (Student's t-test, unpaired, two-tailed). Average trace of time-dependent changes in ER- (D) and mitochondrial Ca2+ (E) in control and INS-1E cells expressing either GPx8C79S or GPx8C79S/C108A upon perifusion with PA. Data are means ± SEM of 3 independent experiments.

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Activity Assay, Mutagenesis, Western Blot, Expressing, Stable Transfection, Transfection, Control, Incubation, MTT Assay, Two Tailed Test

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 8 Summary illustration showing the role of luminal H2O2 in the dysregulation of ER Ca2+ homeostasis unteder lipotoxic conditions. Elevated palmitate concentrations contibuted to increased luminal H2O2 levels that promoted ER Ca2+ depletion by inhibiting SERCA pump actvity. Excessive ER Ca2+ effux affected mitochondrial function manifested by depolarized mitochondrial membrane potential and lowered ATP production. Scavenging of luminal H2O2 by ER-specific antioxidative enzyme GPx8 attanuated palmitate-induced pertubations of intraorganellar Ca2+ dynamics preserved insulin synthesis and mitochondrial function under lipotoxic conditions.

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Membrane