Figure S1.

TMEM106B supports SARS-CoV-2 infection, and TMEM106B-specific monoclonal antibodies are internalized into cells, related to Figures 1 and 2

(A) Confirmation of TMEM106B and ACE2 knockout in monoclonal NCI-H1975 cell lines generated by CRISPR-Cas9. For each sgRNA, the target sequence is shown. The cut site is indicated by an arrowhead, and the protospacer adjacent motif (PAM) is underlined. Wild-type (WT) sequences of the corresponding exons were determined by Sanger sequencing and are presented with chromatograms. Sequences of knockout cells were determined by next-generation sequencing. For each detected sequence variant, the detection frequency and the type of mutation are shown.

(B) NCI-H1975 cells expressing sgRNAs targeting ACE2 (monoclonal) or both ACE2 and TMEM106B and infected with SARS-CoV-2 Belgium/GHB-03021/2020. Viral RNA in cells was measured by qPCR at the indicated time points (n = 8 wells examined over two independent experiments.). p values for differences between ACE2^KO and ACE2^KO/TMEM106B^KO on day 1 were calculated using Mann-Whitney test with Holm-Šídák correction for multiple comparisons. ^∗∗∗0.0001 < p < 0.001.

(C) Huh7 cells transduced with sgRNAs targeting ACE2 and with cDNA encoding luciferase (Luc) or TMEM106B and infected with SARS-CoV-2 Belgium/GHB-03021/2020. Cells were stained for nucleocapsid after 48 h. Infected cells were quantified by high-content imaging analysis (n = 8 wells examined over two independent experiments). Data were analyzed using two-sided unpaired t test with Welch’s correction. Data are mean ± SEM. ^∗∗∗∗p < 0.0001.

(D) Binding of TMEM106B-specific monoclonal antibodies to A549 cells stably overexpressing TMEM106B. Cells were incubated with antibody at the indicated concentrations and stained with Alexa Fluor 647-labeled anti-human IgG. The geometric mean fluorescence intensity (GMFI) compared with an hIgG1 isotype control is shown.

(E) Binding of Ab09 to recombinant AviHis-TMEM106B-luminal domain (LD) was confirmed by surface plasmon resonance. Ab09 bound to recombinant AviHis-TMEM-LD that was captured by immobilized rabbit anti-Avi antibody. This confirmed that the epitope bound by Ab09 was intact on the recombinant protein.

(F) WT and TMEM106B^KO NCI-H1975 cells incubated with Ab09 for 2 h at ambient temperature to assess antibody internalization. Extracellular Ab09 was stained on live cells (green), followed by fixation, permeabilization, and staining of both extracellular and intracellular Ab09 (red) and nuclei (blue). The presence of red foci in WT cells indicates the endocytic uptake of anti-TMEM106B. Scale bars, 20 μm.

(G) NCI-H1975 cells incubated with Ab09 for 50 min at ambient temperature and stained for Ab09 (green), LAMP-1 (red), and nuclei (blue). Four representative images are shown. Scale bars, 10 μm.