Panoply™ Human HSPB1 Knockdown Stable Cell Line

Here, researchers explored the role of heat shock protein family B (small molecule) member 1 (HSPB1) in regulating ferroptosis in glioma cells and investigated its potential molecular mechanisms. The results showed that HSPB1 was significantly overexpressed in glioma cell lines. Knockdown of the HSPB1 gene significantly inhibited the proliferation and invasion of U251 cells, while simultaneously promoting ferroptosis by increasing intracellular Fe2+ levels and lipid peroxidation. BAG3 was identified as a downstream target of HSPB1. Silencing the BAG3 gene reproduced the anti-tumor and pro-ferroptosis effects of HSPB1 gene knockdown. Furthermore, BAG3 overexpression partially reversed the effects of HSPB1 gene silencing, confirming the role of BAG3 in the HSPB1-mediated ferroptosis pathway. These studies suggest that HSPB1 inhibits ferroptosis and promotes glioma cell survival, at least in part, by upregulating BAG3. Targeting the HSPB1-BAG3 axis may represent a novel therapeutic strategy and prognostic assessment method for glioma treatment.

In HSPB1 knockdown cells, GSH levels were significantly decreased (Figure 1A) and GPX activity was significantly reduced (Figure 1B), while reactive oxygen species (ROS) levels (Figure 1C), Fe²⁺ levels (Figure 1D), and MDA levels (Figure 1E) were increased. Western blot analysis showed that HSPB1 silencing decreased GPX4 expression while upregulating transferrin receptor protein 1 (TFR1) expression (Figure 1F). Furthermore, enhanced BODIPY™ 581/591 C11 fluorescence was observed in HSPB1 knockdown cells, indicating increased lipid peroxidation (Figure 1G). Significantly increased LDH release (Figure 1H) and decreased cell viability (Figure 1I) further confirmed enhanced ferroptosis activity. RT-qPCR analysis showed that knockdown of HSPB1 expression also inhibited the expression of Nrf2 and HO-1 (Figure 1J), two proteins that are components of the Nrf2/HO-1 antioxidant pathway, which is known to protect cells from ferroptosis. Taken together, these findings indicate that HSPB1 downregulation promotes ferroptosis in U251 cells by regulating iron metabolism, oxidative stress, and inhibiting antioxidant defense mechanisms.

Figure 1. The effect of HSPB1 on ferroptosis in U251 cells. (Li Q, et al. 2025)