Birnbaumet al.,Science 367 ,51–59 (2020) 3 January 2020 7of9
Fig. 5. Reduced Kelch13 activity leads
to ART resistance.(A) Heatmap of DIQ-BioID
data of the four Kelch13 and the two
Kelch13C580Yexperiments [hits with FDR
< 1% in at least two of four (Kelch13) or
two (Kelch13C580Y) experiments]. Color
intensities: 2nd to 98th percentile of log 2
rapalog/control normalized ratios of all
proteins in this analysis. NaN (data S3) values
in gray. Rows were clustered and gene
identifiers were color coded as in Fig. 2C.
(B) Immunoblot-based quantification of
wtKelch13 (K13wt) and Kelch13C580Y
(K13C580Y) levels in parasites expressing the
otherwise-identical GFP-fused protein from
the endogenous locus. (Left) An example band
pair with Kelch13 levels relative to the loading
control (aldolase) shown as bars below the
blot (axis shows intensity ratio). (Right)
K13C580Ylevels compared with K13wt (n=3
independent experiments; each experiment is
shown by a point that derives from the
average of a serial dilution of K13wt and K13C580Yrun on the same gel and normalized relative to the respective aldolase control). (CandD) Quantification of Kelch13 foci
fluorescence intensity in trophozoites (n= 105, 102 foci) and rings (n= 47, 51 foci) in K13wt and K13C580Yparasites, respectively. Data pooled from three independent
experiments; percent reduction of the mean is indicated. Example fluorescence images of ring stages are shown. (E) Fluorescence microscopy images of parasites
with endogenous Kelch13C580Yepisomally expressing the construct indicated (PSR, plasmodium-specific region; L, linker) and RSA with the respective cell lines (right).
Cell lines expressing mChe-K13wt and mChe-K13C580Ywere imaged after smearing, mChe-K13DPSR live. Size bar, 5mm. (F) Model of resistance and summary of results of this
study. Asterisk indicates only in ring stages; nd, not determined (see also table S1). Bars show SD, mean is indicated. Unpaired, two-tailedttest with Welch's correction.Pvalues
are indicated [in (E), only for selected pairs]; ns, not significant.
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