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Supplemental material, sj-docx-1-npx-10.1177_1934578X211053202 for A Novel Trimeric Triterpene From Chisocheton ceramicus Miq. by Alfarius Eko Nugroho, Marika Okabe, Yusuke Hirasawa and Chin Piow Wong, Toshio Kaneda, Takahiro Tougan, Toshihiro Horii, Hiroshi Morita in Natural Product Communications

Additional file 1: Fig. S1. Schematic illustration of the two procedures for serum preparation. Fig. S2. Serum proteins in RBCs infected with rodent malarial parasite in vivo. Confocal microscopy images showing the localization of vitamin K-dependent protein S (protein S), prothrombin, and vitronectin (red), and DNA (blue) in the RBCs of mice infected with the rodent malarial parasite Plasmodium yoelii in vivo. All samples were fixed with the methanol fixation method. Primary [anti-protein S pAb, anti-prothrombin pAb, anti-vitronectin pAb, or rabbit IgG antibody (control; Catalogue number: 30000-0-AP from Proteintech, Rosemont, IL, USA)] and secondary (anti-rabbit IgG-Alex Flour594) antibodies were suitably diluted (1:200) and used. Images were captured using a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan). “Rabbit IgG” refers to the isotype control antibody. Arrows and arrowheads indicate the iRBCs and non-iRBCs, respectively. The scale bar represents 5 µm. Fig. S3. Serum proteins in RBCs infected with the CDC1 and W2 strains. (a) CDC1 strain. (b) W2 strain. These strains were cultured under the same conditions as those for the 3D7 strain. (Left panels) Serum proteins in the lysate of iRBCs cultured in NCS- or PDS-containing medium. Samples were collected using the precipitation method. (Right panels) Serum proteins in NCS and PDS. The asterisk indicates the putative degradation product. Fig. S4. Confirmation of the generation of Factor Xa for coagulation. Western blotting analysis of Factor Xa, the activated form of Factor X. No signal corresponding to Factor Xa (estimated molecular weight of approximately 28.5 kDa) was detected by anti-Factor Xa pAb (ab111171; Abcam) even when the blot was overexposed. The intensity of the image of Factor X shown in Fig. 5c (i) has been increased. The asterisk indicates the putative degradation product.

Additional file 1: Fig. S1. 1H- and 13C-NMR Spectra of ITT-001. Fig. S2. 1H- and 13C-NMR Spectra of ITT-002. Fig. S3. 1H- and 13C-NMR Spectra of ITT-003. Fig. S4. 1H- and 13C-NMR Spectra of ITT-004. Fig. S5. 1H- and 13C-NMR Spectra of ITT-005·HCl. Fig. S6. 1H- and 13C-NMR Spectra of ITT-006. Fig. S7. Kaplan–Meier curve of representative control groups. Group denoted as “#3” is control group (black line) in Fig. 5.

Additional file 2: Table S1. Parasitaemia of each parasite culture treated with the anti-malarial drugs. Table S2. Growth inhibition rate of parasites treated with the anti-malarial drugs.

Additional file 1: Fig. S1. Representative M scattergram of in vitro cultured sample. The horizontal and vertical axes indicate intensities of side fluorescent light (SFL, which corresponds to DNA content) and forward scattered light (FSC, indicating size of iRBCs), respectively. The colours indicate the following: red, ring-form; orange, trophozoite; purple, schizont; and blue, polychromatic RBC. The colours were assigned based on the default setting of the XN-30 analyzer. The scattergram was cited from Fig. 1a(i), DMSO). Fig. S2. Validation in the assay system. (a and b) 24 h, (c and d) 48 h. (a and c) The scatter-plot of the growth inhibition rate. The growth inhibition rate was calculated based on SCHZ-RBC% at 24 h and MI-RBC% at 48 h (see also Methods). The colours indicate the following: blue, 0.5% DMSO; dark red, positive control (5 µM artemisinin); and dark blue, negative control (saline). (b(i) and d(i)) The growth inhibition rate. (b(ii) and d(ii)) The values of validation indices. Abbreviations are as follows: CV %, coefficient of variation; S/B, signal-to-background ratio; and S/N, signal-to-noise ratio. Fig. S3. M scattergrams of the effective compounds, related to Figs. 3 and 4 and Table 1. (a) Type I, (b) Type II, (c) Type III, (d) Type IV. The left and right panels indicate scattergrams at 24 and 48 h, respectively. *, **, and, ** represent effective compounds described in Tong et al. [19] and Dennis et al. [20], and the reference compound mefloquine, respectively. The colours indicate the following: red, ring-form; orange, trophozoite; purple, schizont; and blue, polychromatic RBC. The colours were assigned based on the default setting of the XN-30 analyzer; however, these may be misclassified after compound treatment as described in the Discussion. Fig. S4. Microscopic images of parasites treated with anti-malarial drugs, related to Fig. 1. (a) ART, (b) CQ, (c) DMSO. Sixteen representative images were randomly selected. Scale bar represents 5 µm. Fig. S5. Two possibilities for the generation of a Type II outcome. Possibility 1: The test compound competed with the DNA staining dye in Fluorocell M, as insufficiently stained DNA are likely to show low DNA content. Possibility 2: The test compound fragmented genomic DNA and the DNA was flowed out; the efflux of fragmented DNA reduced the DNA content. Fig. S6. Microscopic images of Type IV outcome, related to Figs. 3 and 4 and Table 1. Typical microscopic images of parasites treated with MMV026550, MMV011765, MMV024443, or MMV030734 after 48 h of incubation. Scale bar represents 5 µm.

Additional file 1: Figure S1. Analysis of data obtained using the XN-30 analyzer on blood samples from healthy non-infected mice, related to Fig. 1. Figure S2. M scattergrams re-analysed using the Flowing software after parasite infection. Figure S3. Comparison between parasitaemias determined using the XN-30 system and microscopy. Figure S4. Morphological evidence of the high MCV and MPV values in infected blood samples by microscopy. Figure S5. M scattergrams re-analysed using the Flowing software after treatment with artemisinin. Figure S6. Re-analysed scattergrams of mouse blood samples infected with parasites.

Additional file 1: Figure S1. Capability of the XN-30 analyzer equipped with the default algorithm. Figure S2. Representative scattergrams from the XN-30 analyzer, related to Fig. 2b.

Additional file 2: Table S1. Data of false-positive iRBCs and HJB-RBCs in non-infected mice. Table S2. Comparison of parasitaemias obtained using the XN-30 system and microscopy, related to Fig. 3. Table S3. Comparison of parasitaemia, related to Fig. 4a. Table S4. Sequential analysis of RBC count, related to Fig. 4b(i). Table S5. Sequential analysis of WBC count, related to Fig. 4b(ii). Table S6. Sequential analysis of PLT value count, related to Fig. 4b(iii). Table S7. Sequential analysis of HCT value, related to Fig. 4b(iv). Table S8. Sequential analysis of MCV value, related to Fig. 4b(v). Table S9. Sequential analysis of MPV value, related to Fig. 4b(vi). Table S10. Sequential analysis of parasitaemia and the parameters after treatment with artemisinin, related to Fig. 5.

Additional file 2: Table S1. Counts and parasitaemias obtained from the XN-30 analyzer, related to Fig. 2a. Table S2. Counts and parasitaemias obtained from the XN-30 analyzer, related to Fig. 2b. Table S3. Confirmation of gametocytogenesis by microscopy. Table S4. Counts and parasitaemias obtained from the XN-30 analyzer, related to Fig. 2c. Table S5. Parasitaemias obtained from the XN-30 analyzer and microscopy, related to Fig. 3. Table S6. Counts and parasitaemias obtained from the XN-30 analyzer, related to Fig. 4 and Table 1. Table S7. Counts and parasitaemias obtained from the XN-30 analyzer, related to Fig. S2.