Fig 2: miR221 specifically regulates autophagy by targeting LAMP2 expression. (A) Bioinformatics online analysis of the predicted miR221 binding sites of LAMP2. (B) Dual-luciferase reporter assay was performed to detect fluorescence activity of LAMP2. (C-E) Expression of LAMP2 was detected via reverse transcription-quantitative PCR or western blotting analyses in LX2 cells transfected with miR221 mimics or miR221 inhibitors. *P<0.05 vs. NC group. miR, microRNA; LAMP2, lysosome-associated membrane glycoprotein 2; NC, negative control; wt, wild-type; mut, mutant; CTL, control.

Fig 3: Lamp2a modulates viral RNA level in SARS-CoV-2-infected cells. (A) SARS-CoV-2 RNA level (normalized to that of RP) in culture medium of Vero E6 cells transfected with the indicated plasmids and infected with SARS-CoV-2 for 48 h. Data are the mean ± SEM of triplicate samples. P values were calculated using a two-tailed Student t test. (B) Intracellular level of SARS-CoV-2 RNA (normalized to that of RP II) in Vero E6 cells transfected with the indicated plasmids and infected with SARS-CoV-2 for 48 h. Data are the mean ± SEM of triplicate samples. P values were calculated using a two-tailed Student t test. (C) Western blot detection of Lamp2a protein level (using anti-HA antibody [upper panel] and anti-Lamp2 antibody [middle panel]) and GAPDH protein level (lower panel) in pCDNA3-transfected or pcDNA Lamp2a-transfected Vero E6 Cells. (D) Western blot detection of Lamp2b protein level using anti-Lamp2 antibody (upper panel) and GAPDH protein level (lower panel) in pCDNA3-transfected or pcDNA Lamp2b-transfected Vero E6 cells. (E) Western blot detection of Lamp2c protein level using anti-Lamp2 antibody (upper panel) and GAPDH protein level (lower panel) in pCDNA3-transfected or pcDNA Lamp2c-transfected Vero E6 Cells. (F) Western blot detection of Lamp1 (upper panel) and GAPDH (lower panel) protein levels in Huh7 cells transfected for the indicated periods with nontargeting siRNA (NT) or Lamp1 siRNA. (G) Level of SARS-CoV-2 RNA (normalized to that of RP) in culture medium of Huh7 cells transfected for 72 h with Lamp1 siRNA and infected with SARS-CoV-2 for 48 h. Data are the mean ± SEM of triplicate samples. (H) Level of SARS-CoV-2 RNA (normalized to that of RP II) in culture medium of Huh7 cells transfected for 72 h with Lamp1 siRNA and infected with SARS-CoV-2 for 48 h. Data are the mean ± SEM of triplicate samples.

Fig 4: miR221 regulates TGF-β1-induced HSC activation through inhibiting autophagy by targeting LAMP2. TGF-β1 activated HSCs, and simultaneously induced HSC autolysosome dysfunction. The upregulation of miR221 resulted in decreased LAMP2 expression and inactivation of autophagy-lysosomal pathway in TGF-β1-treated LX2 cells. miR221 could induce hepatic fibrosis via mediating autophagy by targeting LAMP2. miR, microRNA; HSC, hepatic stellate cell; TGF-β1, transforming growth factor-β; LAMP2, lysosome-associated membrane glycoprotein 2.

Fig 5: TRIB2 regulates Ub levels via the proteasome.a Ub in the control, Bel-7402 and SMMC-7721 cells with TRIB2 knocked down or overexpressed. b Schematic representation of Ub homeostasis. c CHX chase experiments of Ub in the control and Bel-7402 cells with TRIB2 knocked down. The relative protein levels of conj & poly and mono Ub were normalized to those of GAPDH, respectively, and the “0 h” point was arbitrarily set to 100%. d–f ALP did not participate in the regulation of Ub levels by TRIB2. Ub in the Bel-7402 cells with or without TRIB2 knocked down after treatment with the ALP inhibitors 3-MA (5 mM for 24 h) and CQ (20 µM, 24 h) (d), ATG5 knockout (e), or ATG7 knockdown (f). g Ub in the lysosomes isolated from the same cells as shown in a, as measured by IB. The relative protein levels of conj & poly Ub and mono Ub were normalized to those of LAMP2 as calculated by ImageJ software and indicated just below the blots. h, i TRIB2 did not regulate Ub levels through DUBs. Control and Bel-7402 cells with or without TRIB2 knocked down or overexpressed were treated with DMSO, VLX1570 (20 µM, 24 h), and EOAI (600 nM, 24 h), respectively (h), or in the presence or absence of UCH37 and USP14 knocked down (i). j, k TRIB2 regulated Ub levels via the proteasome. Ub levels at WCL (j) and in proteasome (k) were measured in the same cells shown in a and treated with DMSO or MG132 (8 µM, 12 h). The samples in k derive from the same experiment and the blots have been processed in parallel using control beads. The levels of conj & poly Ub were normalized to that of GAPDH at WCL (j), while normalized to that of PSMB3 in the isolated proteasome (k), and the data are graphed below the blots. Data were analyzed by one-way ANOVA and expressed as mean ± SD. ***P < 0.001; ****P < 0.0001. Images of all the immunoblots are representative of three independent experiments. The relative protein levels of the conj & poly Ub and mono Ub were normalized to those of GAPDH as calculated by ImageJ software and indicated just below the blots (a, d–f, h, i).