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. 2018 Apr 30;38(10):e00608-17. doi: 10.1128/MCB.00608-17

FIG 11.

FIG 11

Accumulation of LC3 in the nucleus in cells positive for IRS-1 nuclear structures is associated with inhibition of autophagy. (A) Western blot analysis of nuclear extracts from HeLa/EV and HeLa/CL006 cells 0, 2, and 4 h following amino acid starvation. The last lane in the blot represents the cytosolic fraction from HeLa/CL006 cells without amino acid starvation (control). The blots were probed with anti-LC3 (Cell Signaling Technology) and anti-p62(SQSTM1) (Santa Cruz). Anti-histone H3 (Cell Signaling Technology) and anti-GAPDH (Santa Cruz Biotechnology) were used as nuclear and cytosolic markers, respectively. (B) Confocal images of HeLa cells cotransfected with the pALS-NLS-IRS-1/mycTag plasmid and the LC3-GFP baculovirus vector. IRS-1 nuclear structures (red fluorescence) were immunolabeled with mouse monoclonal anti-myc tag antibody (catalog number sc-40; Santa Cruz Biotechnology). Both nuclear LC3 and LC3 within autophagosomes are labeled with GFP. Colocalization between nuclear IRS-1 and nuclear LC3 is indicated by an arrow, and cells with autophagosomes are indicated by a white asterisk. Nuclei were counterstained with DAPI (blue fluorescence). (C) Drug resistance detected in tumor cells positive for IRS-1/LC3 nuclear structures. Temozolomide (TMZ)-sensitive LN-229 human glioblastoma cells were transfected with NLS–IRS-1–GFP(HA)/MitoRed and sorted to obtain three mixed populations, expressing low (L), medium (M), and high (H) levels of the transgenes (NLS–IRS-1–GFP and MitoRed). Resistance to temozolomide was tested in monolayer cultures in the presence of 10% FBS with or without 250 μM TMZ. LN-229 cells transfected with an empty vector were used as a control. In both panels, data represent average values ± standard deviations (n = 3). * indicates statistically significant differences between HeLa/EV and HeLa/CL006 cells or between LN-229/EV and LN229/NLS-IRS-1 cells; ** indicates statistically significant differences between LN-229/NLS-IRS-1(low) and LN-229/NLS-IRS-1(high) cells. The inset shows cell sorting and confocal analyses of LN-229 cells expressing the NLS–IRS-1–GFP(HA)/MitoRed transgene. Cells with low, medium, and high levels of the transgene (NLS–IRS-1–GFP) were selected and used for the TMZ resistance assay.