Supplementary Materials

A human apolipoprotein L with detergent-like activity kills intracellular pathogens

Ryan G. Gaudet, Shiwei Zhu, Anushka Halder, Bae-Hoon Kim, Clinton J. Bradfield, Shuai Huang, Dijin Xu, Agnieszka Mamiñska, Thanh Ngoc Nguyen, Michael Lazarou, Erdem Karatekin, Kallol Gupta, John D. MacMicking

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Figs. S1 to S14
  • Table S2
  • Captions for movies S1 to S11
  • Caption for table S1
Table S1
Genome-wide CRISPR/Cas9 screen and RNAseq results. Shown are gene-level MAGeCK P values depicting enrichment in HR versus SR FACS sorted Stm-infected HeLa cells in the presence or absence of IFN-γ. RNAseq analysis depicting the fold induction (relative to untreated) of each gene in the presence of IFN-γ and Stm is also shown.

Images, Video, and Other Media

Movie S1
Bacterial targeting by APOL3. HeLa cell expressing APOL3mnGFP infected with StmRFP. Imaging was initiated 45 min post infection. Images are widefield maximum intensity projections from 5 μm stacks and were bleach corrected using Fiji.
Movie S2
APOL3 mobilization by sterile endosomal damage. HeLa cell expressing APOL3mnGFP imaged immediately after triggering endosomal damage with 1 mM LLOMe. Images are widefield maximum intensity projections from 5 μm stacks and were bleach corrected using Fiji.
Movie S3
Stm IM damage triggered by APOL3 in situ. IFN-γ treated HeLa cell expressing APOL3RFP and infected with Stm-minDmnGFP initiated 45 min post infection. Image is a widefield maximum intensity projection from 5 μm stacks and was bleach corrected using Fiji. Constitutive minDmnGFP expression (used here) results in elongated Stm.
Movie S4
Stm IM damage triggered by APOL3 in situ. A second example of an IFN-γ treated HeLa cell expressing APOL3RFP and infected with Stm-minDmnGFP initiated 45 min post infection. Image is a widefield maximum intensity projection from 5 μm stacks and was bleach corrected using Fiji. Constitutive minDmnGFP expression (used here) results in elongated Stm.
Movie S5
Bacterial penetration of APOL3 in IFN-γ activated cells. 360°C rotation of a 3D surface rendering (Fig. 2f) generated from structured illumination microscopy (SIM) of immunolabeled HA-APOL3 and LPS 150 min post infection in IFN-γ activated cells.
Movie S6
Clearance of APOL3 targeted bacteria from IFN-γ activated cells. IFN-γ treated HeLa cell expressing APOL3mnGFP and infected with StmRFP. Images are widefield maximum intensity projections from 5 μm stacks and were bleach corrected using Fiji. APOL3 coated bacteria (denoted by arrows in first frame) fade from view whereas untargeted bacteria persist.
Movie S7
Stm IM damage triggered by APOL3 in vitro. Stm expressing minDmnGFP were extracted from LLOMe-treated IFN-γ activated ΔAPOL3 cells and treated as mock control. Bacteria were immobilized on agarose pads and wide-field imaging initiated after ~2 min. Images were taken every 12 seconds. scale bar 2 μm.
Movie S8
Stm IM damage triggered by APOL3 in vitro.Stm expressing minDmnGFP were extracted from LLOMe-treated IFN-γ activated ΔAPOL3 cells and treated with CCCP — a known disrupter of IM membrane potential. Bacteria were immobilized on agarose pads and wide-field imaging initiated after ~2 min. Images were taken every 12 seconds. scale bar 2 μm..
Movie S9
Stm IM damage triggered by APOL3 in vitro. Stm expressing minDmnGFP were extracted from LLOMe-treated IFN-γ activated ΔAPOL3 cells and treated with 5 μM rAPOL3. Bacteria were immobilized on agarose pads and wide-field imaging initiated after ~2 min. Images were taken every 12 seconds. scale bar 2 μm.
Movie S10
Permeation of GUVs by APOL3. Single plane from confocal microscopy of GUV's composed of DOPC/DOPG (80:20) incubated in DylightTM 488 free acid (indicator of permeability) were treated with 568-labelled rAPOL3 and imaged live. See movie S11 for 568-rAPOL3 channel alone. Arrow indicates rAPOL3-triggered membrane blebbing occurring concomitantly with GUV permeabilization.
Movie S11
Permeation of GUVs by APOL3. Single plane from confocal microscopy (GUVs described in movie S10) showing the 568-rAPOL3 channel alone.