Fig. 4: βA1-crystallin regulates STAT3 nuclear localization.

a Representative western blot and b, c densitometry graphs showing decreased phosphorylation of STAT3 at tyrosine 705 (p-STAT3^Y705) in βA1 KD astrocytes b untreated or c treated with 30 mM mannitol or high glucose (HG; 30 mM) for 6 h, relative to WT cells (c). βA3 KO cells did not show such changes (a–c); n = 4. **P < 0.01. d, e Overexpression of βA1-crystallin (using βA1-mCherry construct) in βA1 KD astrocytes or treatment with 10 μM PTP1B inhibitor (MSI-1436) for 1 h, prior to HG (30 mM) exposure for 6 h rescued the levels of p-STAT3^Y705, as compared to βA1 KD cells transfected with blank-mCherry construct. βA1-crystallin overexpression was confirmed by mCherry western blot. f Western blot and g densitometry graph showing decrease in p-STAT3^Y705 expression in WT cells infected with Adenovirus-PTP1B overexpression construct or Cryba1 shRNA for 48 h, followed by HG treatment. h Cryba1 knockdown was confirmed by qPCR, which showed about 70% downregulation compared to control. n = 4. *P < 0.05, **P < 0.01. i, j Representative images from live-cell confocal microscopy of human iPSC-derived astrocytes after overexpression of i βA1-mCherry or j βA3-mCherry constructs and k quantitative assessment of nuclear translocation, showing nuclear localization of βA1-crystallin in these cells (i, k), whereas βA3-crystallin construct transfected cells showed less nuclear localization (j, k). Scale bar, 10  μm. **P < 0.01 (n = 16).