Quantitative analysis of proteostasis disruption following siRNA-mediated HSP70 depletion in HeLa cells.

Introduction: Heat shock protein 70 (HSP70) is a central molecular chaperone that maintains proteostasis by supporting protein folding, preventing protein aggregation, and coordinating cellular stress responses. Dysregulation of HSP70 has been associated with cancer progression, neurodegenerative disorders, and aging-related proteotoxicity. However, many experimental approaches rely on chemical inhibition or stress-inducing conditions that may introduce additional confounding effects. This study aimed to investigate the proteostatic consequences of HSP70 depletion in the absence of stress-inducing chemical inhibitors. Methods: A rapid, quantitative workflow was developed to assess chaperone function and proteostasis in cultured cells. HSP70 was depleted in HeLa cells using siRNA-mediated gene silencing. Knockdown efficiency was evaluated at the transcript and protein levels using quantitative polymerase chain reaction (qPCR) and Western blotting, respectively. Flow cytometry was used to quantify ProteoStat-detected misfolded/aggregated protein burden and total cellular fluorescence from a G3BP1-GFP reporter. Results: siRNA-mediated HSP70 depletion resulted in a greater than 90% reduction in HSP70 transcript and protein levels. HSP70-depleted cells exhibited a 1.8-fold increase in ProteoStat fluorescence, indicating an increased burden of misfolded/aggregated proteins, and a 2.9-fold increase in total cellular G3BP1-GFP fluorescence. Because flow cytometry does not resolve intracellular localization or puncta formation, the increased G3BP1-GFP fluorescence should be interpreted as a surrogate reporter response and does not constitute direct evidence of stress granule biogenesis. Discussion: These findings are consistent with impaired proteostasis following acute HSP70 depletion and demonstrate the potential utility of an inhibitor-independent workflow for evaluating cellular responses associated with targeted chaperone depletion. Orthogonal spatial validation using immunofluorescence or confocal microscopy is required to determine G3BP1 localization and confirm stress granule formation. In addition, experiments using multiple independent siRNA duplexes will be necessary to establish target specificity and strengthen the mechanistic interpretation of the observed proteostatic response.