Fig 1: LAL is a transcription target of p53.(A) Previous known p53 response-element (p53–RE) consists of two copies of the palindromic half-site RRRCWWGYYY where each p53 monomer binds five nucleotides (top). A highly p53RE-like sequence was found in LAL promoter region in both human and mouse (bottom). (B) Chip-qPCR assay using IgG or p53 antibody from control, 24 hr fasted, and refed mouse primary adipocytes. (C) Luciferase reporter constructs were generated by using 1 kb wild-type (WT) or p53-RE deleted (?p53RE) LAL promoter sequence. (D) 3T3-L1 adipocyte were transiently transfected by electroporation with constructs from C together with pcDNA3.1-p53 construct. After 48 hr, cells were harvested in the reporter lysis buffer. Luciferase activity in cell lysates was assayed as described under ‘Experimental Procedures’ and normalized by protein concentrations. Data are presented for triplicate samples as mean ± S.D. (error bars). **, p<0.01. Figure 5—source data 1.An Excel sheet with numerical quantification data.
Fig 2: Deletion of p53 suppresses adipocyte lipid metabolism through the down-regulation of lysosomal acid lipase.(A) Long-term lipolysis activities were measured in Iso-stimulated p53-knockout (KO) and wild-type (WT) control adipocytes. (B) LAL mRNA expression levels were examined in the samples from the end time point (36 hr) of A (C) Protein expression levels of LAL, ATGL, and actin were examined by western blots in control, LAL, ATGL, and LAL/ATGL double RNAi knockdown cells. (D) 3T3-L1 adipocytes from C were cultured with or without ISO stimulation (in PBS +1% BSA) for 18 hr and the concentrations of fatty acid in the medium were measured. (*p<0.05). (E) LAL was overexpressed in WT and p53–KO 3T3-L1 adipocytes, and (F) long-term lipolysis (18 hr) activities were measured by determining FFA levels. (*p<0.05, **p<0.01). Figure 4—source data 1.An Excel sheet with numerical quantification data.
Fig 3: LAL protein undergoes ubiquitination-mediated inactivation in an in vitro model of NAFLD.Huh7 cells were cultured in high-glucose/high-lipid (HGHL) medium for 4 (A) or 8 days (B–D) or with proteasome inhibitor MG132 (10 µM) for 6 h (E, F). A Evaluation of LAL protein, ubiquitinated proteins (Ub-prot) and cathepsin D through western blot analysis. TOMM20 was used as loading control. B–D Analysis of LAL ubiquitination by confocal microscopy. Ubiquitinated proteins (red) and total LAL protein (individuated by antibody against C-term) (green) were shown (B), and their abundance (C) and colocalization (D) were evaluated. E Evaluation of LAL (by different anti-LAL antibodies against C-term or N-term) and ubiquitinated proteins (Ub-prot). Vinculin was used as loading control. F LAL activity determined by spectrophotometric enzymatic assay. G Representative western blot of Huh7 cells expressing HA-tagged ubiquitin. Anti-HA antibody was used to reveal ubiquitinated proteins under 4 days HGHL treatment. Ponceau red staining was used as loading control. H Western blot analysis of LAL by using C-term or N-term antibody on immunoprecipitated HA-ubiquitinated proteins under 4 days HGHL treatment. Immunoblots are representative of one experiment out of three giving similar results. Data are expressed as mean ± SD (*p < 0.05, **p < 0.01, vs CTR; n = 3). Scale bar 25 µm.
Fig 4: LAL protein content and activity is affected in an in vivo model of NAFLD.C57BL/6J mice were fed for 4 and 8 months with high-fat diet (HFD) or normal diet (ND). A Immunohistochemistry analysis of LAL in liver by using different antibodies recognizing total LAL (C-term) or non-ubiquitinated LAL (N-term) and the relative quantification compared to ND. B Evaluation of total LAL protein in liver homogenates by western blot analysis (left panel) and relative densitometric analysis (right panel). Ponceau red staining was used as loading control. C LAL activity determined by spectrophotometric enzymatic assay. D Analysis of LIPA mRNA levels by qPCR. E–G Analysis of LAL localization by confocal microscopy. The lysosomal marker LAMP1 (red) and total LAL protein (C-term) (green) were shown (E) and their abundance (F) and colocalization (G) were evaluated. Representative immunohistochemistry and immunofluorescent images are reported that are from one mouse out of 3 for each group. Data are expressed as mean ± SD (*p < 0.05; **p < 0.01, ***p < 0.005, vs ND; n = 3 each group). Original magnification X100 (A); X400 (E), high-power field X600 (E). Scale bar 250 µm (A); 25 µm (E).
Fig 5: LAL protein accumulates in the extra-lysosomal compartment in an in vitro model of NAFLD.Huh7 cells were cultured in high-glucose/high-lipid (HGHL) medium for 8 days. A–C Analysis of LAL localization by confocal microscopy. The lysosomal marker LAMP1 (red) and total LAL protein (individuated by antibody against C-term) (green) were shown (A) and their abundance (B) and colocalization (C) were evaluated. Scale bar 25 µm. D, E Western blot analysis of LAL C-terminal domain, LDHB, Tubulin and LAMP-1 in in the cytosolic fraction (D) and evaluation of LAL content in the cytosolic fraction by calculating LAL to tubulin ratio through densitometric analysis (E). Representative images from one experiment out of three giving similar results are reported. Data are expressed as mean ± SD (*p < 0.05, **p < 0.01 ***p < 0.001, vs CTR; n = 3).
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