Fig 2: HCV-mediated decrease in LIPC activity modulates triglycerides and promotes virion production.A, quantification of triglyceride levels in cells exogenously expressing LIPC protein, n = 3. B–D, quantification of LIPC activity, protein expression, and mRNA expression in Huh7.5 cells infected with JFH1T. B, representative sample of Western blotting on FP-B labeled enriched fraction and cell lysates. C, densitometric quantification of active LIPC in the FP-B labeled and enriched fraction and its abundance in cell lysates, n = 3. D, qRT-PCR quantification of LIPC mRNA, n = 4. E, qRT-PCR quantification of intracellular HCV RNA in Huh7.5 cells transfected with LIPC siRNA and HCV RNA in cells infected by virus secreted from siRNA-transfected cell, n = 3. F, qRT-PCR quantification of LIPC in Huh7.5 cells transfected with 10 nM LIPC siRNA, n = 3. Values shown are normalized to the naïve or control-transfected sample. Data represent mean values ± SD. *p = 0.05, **p = 0.01. FP-B, fluorophosphonate-biotin; HCV, hepatitis C virus; LIPC, lipase C; qRT-PCR, quantitative RT-PCR.

Fig 3: LIPC activity and expression are downregulated by miR-27b during HCV infection. Quantification of fluorophosphonate (FP) labeling of LIPC, LIPC protein expression, and LIPC mRNA in JHF1T-infected Huh7.5 cells transfected with miR-27b mimic, inhibitor, or their respective negative controls. Results represent signal from mimic or inhibitor-transfected samples normalized to their respective controls. A, representative sample of Western blotting on FP-B labeled enriched fraction and cell lysates. B, densitometric quantification of FP-B labeling of active LIPC detected by Western blotting on the enriched fraction, n = 3. C, densitometric quantification of LIPC protein expression detected by Western blotting, n = 3. D, densitometric quantification of the protein expression of positive transfection control EGFR, n = 3. E, densitometric quantification of the protein expression of HCV CORE protein detected by Western blotting, n = 3. F, qRT-PCR quantification of LIPC mRNA, n = 4. G, qRT-PCR quantification of mRNA of positive transfection control EGFR, n = 4. H, qRT-PCR quantification of HCV CORE mRNA, n = 4. Data represent mean values ± SD. *p = 0.05, **p = 0.01, ***p = 0.001. EGFR, epidermal growth factor receptor; FP-B, FP-biotin; HCV, hepatitis C virus; LIPC, lipase C; qRT-PCR, quantitative RT-PCR.

Fig 4: LIPC activity and expression are down-regulated by miR-27b. Quantification of fluorophosphonate (FP) labeling of LIPC, LIPC protein expression, and LIPC mRNA in Huh7.5 cells transfected with miR-27b mimic, inhibitor, or their respective negative controls. Results represent signal from mimic or inhibitor-transfected samples normalized to their respective controls. A, representative sample of Western blotting on FP-B labeled enriched fraction and cell lysates. B, densitometric quantification of FP-B labeling of active LIPC detected by Western blotting on the enriched fraction, n = 3. C, densitometric quantification of LIPC protein expression detected by Western blotting, n = 3. D, qRT-PCR quantification of LIPC mRNA, n = 4. E, densitometric quantification of the protein expression of positive transfection control EGFR detected by Western blotting, n = 3. F, qRT-PCR quantification of positive transfection control EGFR mRNA, n = 4. Data represent mean values ± SD. *p = 0.05, **p = 0.01, ***p = 0.001 using a two-tailed Student’s t test. EGFR, epidermal growth factor receptor; FP-B, FP-biotin; LIPC, lipase C; qRT-PCR, quantitative RT-PCR.

Fig 5: miR-27b modulation of LIPC expression occurs via modulation of multiple transcription factors.A–C, quantification of changes to LIPC activity and protein expression after treatment of Huh7.5 cells with 75 µM PPAR inhibitor benzamide, or after transfection with miR-27b mimic and treatment with 75 µM PPAR activator bezafibrate. A, representative Western blot, consisting of the Western blots shown in Figure 4, expanded to include benzamide and bezafibrate treatments. B, densitometric quantification of Western blotting against LIPC in the FP-B labeled fraction and in cell lysates, n = 3. C, densitometric quantification of Western blotting against HNF4a isoforms, n = 3. D, qRT-PCR quantification of mRNA expression of LIPC-associated transcription factors in miR-27b mimic versus control-transfected cells, n = 4. Results represent signal from transfected or treated samples normalized to their respective controls. Data represent mean values ± SD. *p = 0.05, **p = 0.01, ***p = 0.001 using a two-tailed Student’s t test. FP-B, FP-biotin; LIPC, lipase C; PPAR, peroxisome proliferator-activated receptor; qRT-PCR, quantitative RT-PCR.