pcDNA3.1(-)

Background
This STAR Protocols article presents a step-by-step method for overexpressing and detecting human asprosin in cultured cells and mice. It avoids unreliable recombinant asprosin by using adenoviral, AAV8, and plasmid vectors for sustained asprosin expression. The protocol includes HEK293T transfection, tail-vein injection in C57BL/6J mice, plasma IgG/albumin depletion, and a custom sandwich ELISA with anti-asprosin monoclonal antibodies. Expected outcomes include measurable asprosin in conditioned media and plasma, with functional validation via increased food intake and body weight.
Key Findings
The protocol establishes reliable overexpression and ELISA-based detection of asprosin without commercial recombinant protein. HEK293T cells transfected with pcDNA3.1-IL2sp-6His-Asprosin secreted approximately 1.5–2.5 nM asprosin into conditioned media 72 h post-transfection, detectable by sandwich ELISA using mouse and rabbit anti-asprosin monoclonal antibodies. In mice, tail-vein injection of Ad5-hFBN1 produced transient asprosin elevation peaking around day 12 and reverting by day 25, accompanied by increased food intake and body weight. AAV8-IL2sp-6His-Asprosin achieved peak expression at 40–60 days, with 2–3 fold higher plasma asprosin and concomitant hyperphagia and weight gain. The custom ELISA reliably detected overexpressed asprosin in IgG/albumin-depleted, concentrated mouse plasma and cell culture media. These results provide a validated alternative to variable recombinant asprosin preparations and inconsistent detection assays.
Contribution
Creative Biogene supplied the empty pcDNA3.1 plasmid (Cat# VET1452) used as the negative control for HEK293T cell transfections. This control enabled direct comparison with pcDNA3.1-IL2sp-6His-Asprosin, validating asprosin overexpression at the protein level and supporting the ELISA-based detection in cultured cells.

Background
This study investigates the role of TXNIP in high glucose (HG)-induced dysfunction of human retinal pigment epithelial (RPE) cells. HG increases TXNIP expression, causing mitochondrial membrane depolarization, fragmentation, and mitophagic flux to lysosomes. Using mt-Keima and LAMP1-mCherry imaging, HG is shown to enlarge lysosomes, inactivate cathepsin L, and activate caspase-1. TXNIP knockdown by shRNA, antioxidant NAC, or Amlexanox prevents mitochondrial fragmentation, mitophagic flux, and lysosome enlargement, indicating that TXNIP mediates deleterious HG effects in RPE that may contribute to diabetic retinopathy.
Key Findings
High glucose (25 mM) significantly upregulates TXNIP at mRNA and protein levels in ARPE-19 and primary human RPE cells compared with low glucose (5.5 mM). TXNIP induction is linked to mitochondrial membrane depolarization, reduced ATP, decreased cell viability, mitochondrial fragmentation, and mitophagic flux to lysosomes. Confocal live-cell imaging with mt-Keima shows an elongated mitochondrial network under low glucose and fragmented mitochondria with enlarged red mt-Keima lysosomes under high glucose. LAMP1-mCherry imaging confirms two- to threefold lysosomal enlargement. High glucose also causes cathepsin L inactivation and caspase-1 activation, suggesting lysosomal destabilization and inflammasome activation. TXNIP knockdown by shRNA prevents mitochondrial fragmentation, mitophagic flux, lysosomal enlargement, and cathepsin L inactivation. The antioxidant N-acetylcysteine and Amlexanox, a TBK1 inhibitor, also block mitophagic flux and lysosome enlargement. These results suggest that TXNIP mediates high glucose-induced mitochondrial damage, mitophagic overload, and lysosome dysfunction in RPE, potentially contributing to diabetic retinopathy.
Contribution
Creative Biogene provided the TXNIP shRNA clones in pcDNA3.1 plasmids used for TXNIP knockdown in ARPE-19 cells. This reagent enabled key loss-of-function experiments in the study showing that TXNIP mediates high glucose-induced mitophagic flux and lysosome enlargement.