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Supplemental Information

Supplementary Images showing raw data for the Western blots

DOI: 10.7287/peerj.preprints.2813v2/supp-1

Combinations and dilutions of the antibodies and their method of generation and checking the specificity

DOI: 10.7287/peerj.preprints.2813v2/supp-2

Quantification data for various drug treatments

S2 (A): Percentage distribution of PfTPxGl in control, drug treated and drug washed out parasites. (B): Percentage of parasites showing intact/collapsed ER-Golgi morphology in drug treated and drug washed out cells. (C): Percentage of parasites showing PfEMP1 and KAHRP distribution in drug treated and drug washed out parasites.

DOI: 10.7287/peerj.preprints.2813v2/supp-3

Effect of Aluminum tetrafluoride (AlF4-) treatment on P. falciparum cultures.

(A) Dose response curve fit for P. falciparum treated at different concentrations of Aluminum tetrafluoride (AlF4-). IC50 value calculated non-linear regression of the sigmoidal dose response equation from OriginPro was found to be 1.23±0.16 µM. 95% confidence interval was found to be ±0.16. Note that no parasite survival was observed at higher AlF4- concentrations of 7.5 and 10 µM at 24 hours. (B) Images showing a normal morphology of P. falciparum treated with Aluminum tetrafluoride (AlF4-) and vineblastine at IC50 concentration of 1.2 µM and 100 nM respectively for 18±2 hours.

DOI: 10.7287/peerj.preprints.2813v2/supp-4

Immunofluorescence images showing PfKAHRP trafficking in control, AlF4--treated and vinblastine treated D10-ACPleader-GFP parasites

In AlF4- treatment, KAHRP trafficking to the RBC cytosol and surface was inhibited in 97% of the parasites 87 parasites analyzed. In vinblastine treated cultures, KAHRP trafficking to the RBC cytosol and surface was inhibited in 98% of the 56 parasites analyzed (See Supplementary Table 2 for quantification). Scale Bar: 10 µm.

DOI: 10.7287/peerj.preprints.2813v2/supp-5

Immunofluorescence images showing PfTPxGl and PfFC (mitochondrial marker protein) trafficking in AlF4-, nocodazole and vineblsatine treated 3D7 parasites

(A) PfTPxGl and PfFC co-localization in AlF4--treated parasites, (B) PfTPxGl and PfFC co-localization in vinblastine-treated parasites, (C) PfTPxGl and PfFC co-localization in nocodazole-treated parasites. In this experiment, PfTPxGl was found to be co-localized with the mitochondrial marker protein PfFC in 40% of the treated parasites suggesting that trafficking of PfTPxGl to the mitochondrion may be partially disrupted by the treatments. Scale Bar: 10 µm.

DOI: 10.7287/peerj.preprints.2813v2/supp-6

Immunofluorescence images showing PfTPxGl trafficking in AlF4-, nocodazole and vineblsatine treated 3D7 parasites

(A) PfTPxGl localization in solvent control parasites, (B) PfTPxGl localization in AlF4--treated parasites, (C) PfTPxGl localization in nocodazole-treated parasites, (D) PfTPxGl localization in vinblastine-treated parasites, (E) PfTPxGl localization in parasites reverted after nocodazole treatment, (F) PfTPxGl localization in parasites reverted after vinblastine treatment. PfTPxGl targeting was disrupted in 97% (35 parasites counted) of nocodazole-treated parasites and in 96% (33 parasites counted) of vinblastine-treated parasites. For AlF4--treatment, 92% of the 72 parasites analyzed showed disrupted PfTPxGl signal. In reversion experiments, localization of PfTPxGl in vinblastine washed out parasites was reverted to the apicoplast in 52% parasites (48 parasites counted), while in nocodazole washed out parasites 49% showed apicoplast localization (59 parasites counted) (See Supplementary Table 2 for quantification). Scale Bar: 10 µm.

DOI: 10.7287/peerj.preprints.2813v2/supp-7

Immunofluorescence images showing PfTPxGl and microtubules in nocodazole-treated D10-ACPleader-GFP parasites

(A) In these experiments, targeting to the apicoplast was inhibited in 97% of the parasites with nocodazole treatment (35 parasites counted), (B) Immunofluorescence images showing PfTPxGl and microtubules in D10-ACPleader-GFP parasites with drug washed out. Reversion of PfTPxGl localization to the organelles and intact microtubular structures observed in parasites in drug washed out medium after nocodazole treatment. In reversion experiment, localization of PfTPxGl in nocodazole washed out parasites was reverted to the apicoplast in 45% parasites (22 parasites counted). Scale Bar: 10 µm.

DOI: 10.7287/peerj.preprints.2813v2/supp-8

Immunofluorescence images showing the endoplasmic reticulum (ER) morphology in AlF4- and vinblastine treated parasites

(A) PfBiP localization in control parasites, (B) ER morphology in AlF4-- treated parasites (C) ER morphology in vinblastine-treated parasites, (D) ER morphology in parasites reverted after vinblastine treatment. Scale Bar: 10 µm.

DOI: 10.7287/peerj.preprints.2813v2/supp-9

Immunofluorescence images showing the Golgi morphology using PfGRASP as a marker protein

DOI: 10.7287/peerj.preprints.2813v2/supp-10

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Rahul Chaudhari conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Vishakha Dey conceived and designed the experiments, performed the experiments, analyzed the data, reviewed drafts of the paper.

Aishwarya Narayan analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Shobhona Sharma conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Swati Patankar conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Human Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The work was approved by the Institute Ethics Committee and Institute Biosafety Committee at Indian Institute of Technology Bombay. Written informed consent was provided by all the blood donors.

Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The work was approved by the Institute Ethics Committee and Institute Biosafety Committee at Indian Institute of Technology Bombay. Written informed consent was provided by all the blood donors.

Data Deposition

The following information was supplied regarding data availability:

The raw data has been supplied in the Figures and Supplemental files.

Funding

This work was supported by grants from Science and Engineering Research Board (SERB, Project File no. SB/YS/LS-354/2013) and Board of Research in Nuclear Sciences (BRNS, Project File no. 2013/37B/18/BRNS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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