Supplemental material for mBio00630-22

SUPPLEMENTAL FIGURES

Autophagy Underlies the Proteostasis Mechanisms of Artemisinin Resistance in P. falciparum Malaria

Ananya Ray,^a Miti Mathur,^a Deepak Choubey,^b Krishanpal Karmodiya,^c Namita Surolia^a,#

a. Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India

b. Life Science Research unit, Persistent Systems Limited, Pune, India

c. Department of Biology, Indian Institute of Science Education and Research, Pune, India

Running Head: Autophagy underlies mechanisms of ART resistance

# Address correspondence to Namita Surolia (N.S.), surolia@jncasr.ac.in

Fig S1 ART induced ER stress activates the UPR pathway. To visualize the expansion and morphology of the parasite ER, PfSec62, a translocon complex component present on the ER membrane was labelled with GFP. (A) Live cell confocal microscopy images of P. falciparum 3D7 parasites overexpressing PfSec62-GFP. ER expansion is observed after 1.5h of incubation with DHA. PfSec62-GFP signal extends into the parasite cytoplasm whereas it localizes to the nuclear periphery in control (untreated) parasites. ER expansion is analysed using GFP fluorescence intensities measured along the white line in the enlarged panel, and were plotted as the line scan graphs. Nucleus was stained using Hoechst. Scale bar: 5 μm. Scatter plot represents the extent of ER expansion as measured by the distance of PfSec62-GFP signal (obtained from line scan graphs of GFP fluorescence intensities) in control and parasites incubated with DHA. N = 25 parasites, n = 3 independent experiments. The data points are expressed as mean ± standard error of the means (SEM). Statistical significance is quantified using unpaired Student’s t-test, **** = P < 0.0001. (B) 3D reconstruction with Z-stacks obtained from confocal images of control and of parasites incubated with DHA overexpressing PfSec62-GFP. Volume of ER is measured through the surface function in Imaris 3.0. (C) Effect of DHA on the phosphorylation status of PfeIF2𝛼.Control and parasites incubated with DHA were harvested. Parasite lysates were subjected to immunoblot analysis and blots were probed with phosphorylated-PfeIF2𝛼 and total PfeIF2𝛼 antibodies. β-Actin was used as the loading control. Fold difference, normalized with respect to control is shown below each blot.

Fig S2 ART increases expression of the autophagy proteins. P. falciparum 3D7 parasites were incubated with either 700nM DHA or starvation media for 1.5h and harvested. 3D7 parasite lysates were subjected to immunoblot analysis and blots were probed with PfATG8 and PfATG18 antibodies as shown in Fig. 1B. Graphs showing the fold changes of PfATG8 (left) and PfATG18 (right) protein expression levels upon incubation with DHA and starvation media. n = 3 independent experiments. The data points are expressed as mean ± SEM. Statistical significance is quantified using unpaired Student’s t-test, *** = P < 0.0005, * = P < 0.05, ns = nonsignificant.