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. 2021 Mar 4;11:599124. doi: 10.3389/fonc.2021.599124

Figure 2.

Figure 2

HMGB1 regulates autophagy levels through AMPK/mTOR signaling. (A) Protein levels of HMGB1 and autophagy makers were determined in two HCC cell lines with a non-targeting control lentivirus-shRNA (control) or three lentivirus-shRNA targeting HMGB1 mRNA (sh1, sh2, and sh3). Related protein levels were quantified and analyzed. (B) Q-PCR experiments were performed to detect mRNA expressions of autophagy markers in both Bel7402 and HCCLM3 cell lines with or without HMGB1 lentivirus-shHMGB1 transfection (shHMGB1). (C) Representative images of immunofluorescence (IF) staining LC3B in Bel7402 control, sh1, and sh2 cells. Scale is 100 μm. Numbers of LC3B puncta in three groups were quantified and analyzed. (D) Representative images of autophagesomes (red arrow) in Bel7402 control, sh1, and sh2 cells. Scale is 2 μm. Numbers of autophagosomes in three groups were quantified and analyzed. (E) Identification of the essential role of AMPK/mTOR signaling in autophagy induction in both Bel7402 shHMGB1 and HCCLM3 shHMGB1 HCC cells by AMPK siRNA transfection in (siAMPK). (F) Identification of HMGB1-mediated regulation of AMPK/mTOR signaling pathway in both Bel7402 and HCCLM3 cells by HMGB1 plasmid transfection (pEnter-HMGB1, 2 μg/ml). Data are means ± SEM from three independent experiments, * means p<0.05, ** means p<0.01, *** means p<0.001 by unpaired student T test.