Fig 2: GalNAc siRNA Conjugates Elicit Functional Intervention in MPCCs(A and B) Secreted AAT (A) and albumin (B) levels determined by ELISA in supernatants of MPCCs treated with 25 nM of GalNAc-conjugated or GalNAc-free AAT siRNA, in free-uptake mode. Values were normalized to untreated cells. Data represent the mean ± SEM (n = 3); each independent experiment was performed with a different hepatocyte donor. See Figure S2B for dose-dependent silencing over time.(C and D) CD81 hepatocyte surface expression in MPCCs 3 days post-treatment with CD81 siRNA GalNAc-conjugated or GalNAc-free control siRNA, or AAT (off-target) GalNAc-conjugated siRNA (25 nM). Representative images of hepatocyte islands immunostained with anti-CD81 antibodies (green) are shown in (C). Nuclei of hepatocytes and fibroblasts are shown in blue. The white dotted line depicts the border of the island of hepatocytes. Scale bar, 50 µm. (D) Graph bars represent mean ± SD of CD81 fluorescence intensity staining per island. p < 0.0001.(E–G) P. falciparum infection in MPCCs treated with 25 nM siRNA 3 days prior to infection. Primaquine treatment (250 nM) was initiated 3 h post-infection and repeated daily. MPCCs were fixed at 4 days post-infection and analyzed by immunofluorescence assays.(E) Representative images of P. falciparum hepatic forms. Parasites were detected using antibodies against the parasitophorous vacuolar membrane P. falciparum exported protein 1 (PfEXP1, red) and the cytoplasmic heat shock protein 90 (PfHSP90, green). Scale bar, 5 µm.(F) Quantification of parasite numbers (EEFs, exoerythrocytic forms) per well (mean ± SEM). p < 0.01.(G) Quantification parasite size distribution (mean ± SD). Data represent one of two independent infections.

Fig 3: The King’s mutation functionally inactivates human A1AT and mouse Serpina1a. Analysis of neutrophil elastase (NE) inhibitory function of supernatants from human A1AT (A) or mouse Serpina1a (B) expressing COS-7 cells. Human and mouse antitrypsin protein concentrations were determined by western blot quantification (see Supplementary Fig. S6A–D) and 10 ng of total protein were used for inhibition of NE. Recombinant human A1AT or mouse Serpina1a protein was included for standardization, whereas chemical NE-inhibitor SPCK and supernatants from untransduced COS-7 cells served as positive and negative controls, respectively. ?RFU was calculated as difference in fluorescence at Ex/Em = 400/505 nm between time points t = 0 min and t = 30 min. All supernatants were analyzed using n = 3 biological replicates × n = 3 technical replicates each. SPCK and recombinant protein controls were analyzed using n = 3 technical replicates. Error bars represent +SD. A full statistical evaluation using one-way ANOVA can be found in Supplementary Table 2.

Fig 4: Serpina1a mutants imitate intracellular distribution of human Z-A1AT. Confocal laser immunofluorescence analysis of COS-7 cells expressing wild type (wt; top row), E337K, H329D or H329D_E337K double mutant Serpina1a (second to fourth row), compared to cells overexpressing normal human M-A1AT or E342K Z-A1AT (fifth and sixth row). Mouse Serpina1a and human A1AT were stained with Alexa Fluor 568 secondary antibody (green) and exposed to 0.6% laser light transmission (LLTM). Mutant-expressing cells were additionally exposed to 0.1% LLTM, as the very strong signal resulted in over-saturation at 0.6%. ER-marker Grp78/BiP was stained with Alexa Fluor 647 secondary antibody (red) and cell nucleus was stained using DAPI (blue). Scale bar: 10 µm.

Fig 5: Serpina1 mutants self-aggregate and cause ER dilation. (A) Native western blot with cell lysates from COS-7 cells expressing wt and mutant Serpina1a. Untransduced COS-7 cells and recombinant Serpina1a served as negative and positive controls, respectively. (B) Quantification of dilated ER in electron microscopy images from wild type Serpina1a and E337K-mutant overexpressing COS-7 cells. Over 27,000 points were analyzed in >400 non-overlapping images and percentage of points in dilated ER was calculated.