Fig 2: Lysosomal targeting of PCSK9-ΔCTD bypasses the need of CTD to induce LDLR degradation.(A-B) HepG2 cells were transfected without (Empty) or with V5-tagged PCSK9 full-length (FL), ΔCTD, CTD alone or PCSK9-TM-CT-Lamp1 chimeras (FL, F379A, ΔCTD, CTD) or Timp1-TM-CT-Lamp1 (herein used as control). Forty-eight hours post-transfection, LDLR, PCSK9 and β-actin (loading control) protein levels were analyzed by IB. Data are representative of at least three independent experiments

Fig 3: LDLR-dependent uptake of Fc(A680)-VH4127 conjugate by pancreatic cancer cells.a Schematic structure of the fluorescent Fc(A680)-VH4127 conjugate. b Kinetic parameters (kon and koff) and equilibrium dissociation constant (KD) of Fc(A680)-VH4127 conjugate. Data are represented as mean ± standard deviation (SD). n = 3 independent experiments. c Flow cytometry analysis of total and internalized Fc(A680)-VH4127 in Ldlr WT and KO PK4A cells with or without DiI-LDL. Data are represented as fold-change ± s.e.m. of the mean of fluorescence intensity (MFI) of Fc(A680)-VH4127 expressed relative to value measured in Ldlr WT cells incubated without DiI-LDL and arbitrarily set to 1000. One-way ANOVA with post-hoc Tukey HSD test, ns: no significant difference, ***p < 0.001. n = 3 independent experiments. d, e Representative confocal microscopy images of Fc(A680)-VH4127 (pseudo-colored red) and DiI-LDL (pseudo-colored green) (d) or LysoTracker blue (e) in live Ldlr WT and KO PK4A cells. ×60 magnification, scale bar: 10 µm. An enlarged merge image of indicated part is provided in inset box. n = 3 independent experiments.

Fig 4: Human LDLR is proteolytically cleaved in its extracellular ligand binding domain by BMP1. (A) Schematic of the domain organisation of LDLR with a C-terminal FLAG tag showing the epitopes detected by the antibodies used in the study, antibody AF2148 (R&D Systems) raised against the entire ectodomain of LDLR, antibody Ab14056 (Abcam) raised against a recombinant protein fragment corresponding to amino acids 29–205 of LDLR) and antibody a-FLAG (Sigma-Aldrich) the anti-FLAG M2 antibody. TM, transmembrane domain; EGF, epidermal growth factor-like domain; F, FLAG epitope. (B) Immunoblot analysis with the indicated antibody of lysates and conditioned media samples from HepG2 cells expressing full-length FLAG-tagged human LDLR. Bands of interest were cropped from western blots of either media or lysate samples using each of the three antibodies. Images from separate western blots were combined but are separated by the dashed black line. Full blot images are presented in the Supplementary western blot dataset. (C) Schematic showing the proposed cleavage of the 160 kDa full-length (FL) LDLR to generate the 36–40 kDa NTF and 120 kDa CTF. (D) Immunoblot analysis of LDLR (antibody AF2148) without and with deglycosylation in liver biopsy samples from three separate individuals. The blot image was cropped to highlight the FL and CTF bands, full blot images are presented in the Supplementary western blot dataset. (E) Immunoblot analysis following incubation of rhLDLR (500 ng) with increasing amounts of rBMP1 at 37 °C for 1 h. (F) Immunoblot analysis following incubation of rhLDLR (500 ng) with rBMP1 (12.5 ng) in the absence or presence of the BMP1 inhibitor UK383367 (10 µM) at 37 °C for 1 h. (G) Immunoblot analysis following pre-incubation of rhLDLR (500 ng) in the absence or presence of LDL (5 µg), RAP (7.14pmol) or UK383367 (10 µM) for 30 min on ice followed by the addition of 12.5 ng rBMP1 and further incubation at 37 °C for 1 h. (H) Densitometric analysis of the Ab14056 immunoblot from (C) to determine the amount of FL and NTF as a percentage of total LDLR, data shown as mean ± SEM, statistical analysis using ANOVA with Tukey post-hoc pairwise analysis *p < 0.05, n = 3. For panels E–G, blot images were cropped to highlight the FL and CTF bands using the AF2148 antibody and the FL and NTF bands using the Ab14056 antibody due to different exposure times for visualisation of the FL and NTF bands. Full blot images are presented in the Supplementary western blot dataset.

Fig 5: PCSK9 regulates lipoprotein-dependent uptake of LTA and LPS by HepG2 cells through LDLR, not LRP1. HepG2 cells were cultured in 20% normal human serum (A,C) or (commercially available) lipoprotein-deficient human serum (B,D) and were pre-treated with 2.5 μg/mL of recombinant human PCSK9 or vehicle control at 6 h before, as well as anti-LDLR, anti-LRP1, or control IgG antibodies at 2 h before treatment with BODIPY 630/650-LTA (10 μg/mL; A,B) or AlexaFluor 488-LPS (2.5 μg/mL; C,D) for 24 h. Data were collected from 4–5 experiments at 24 hours after LPS or LTA treatment using flow cytometry, and are expressed as geometric mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA. MFI, mean fluorescence intensity.