*4.9. Acute Inflammation Induced by TmC4-47.2*

Balb/c mice were intraperitoneally injected with TmC4-47.2 at 10 μg in 500 μL, according to Lima et al. [32]. As a negative control, mice were injected i.p. with PBS. After 2, 16, and 24 h, the peritoneum exudates were harvested for total and differential cell count and protein determination. IL-6, TNF-α, IL-1β, KC, eotaxin, and MCP-1 were analyzed using a specific two-site sandwich ELISA with OpEIA Kits (BD-Pharmingen, San Diego, CA, USA). The total leukocyte count was performed in Turk solution, and for differential counts, neutrophils (Ly6G+), eosinophils (CCR3+), and macrophages (F4/80+) were identified by flow cytometry and based on staining and morphologic characteristics using a light microscope Axio Imager A1 (Carl Zeiss, Germany) with an AxioCam ICc1 digital camera (Carl Zeiss).

#### *4.10. Statistical Analysis*

All values were expressed as mean ± SEM. Experiments using 3 to 5 mice per group were performed independently two times. Parametric data were evaluated using analysis of variance, followed by the Bonferroni test for multiple comparisons. Non-parametric data were assessed using the Mann-Whitney test. Differences were considered statistically significant at *p* < 0.05 using GraphPad Prism (Graph Pad Software, v6.02, 2013, La Jolla, CA, USA).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/toxins14010002/s1. Figure S1: (A) Fractionation process of the *Thalassophryne maculosa* venom. A total of 5 mg of venom was applied to a semi-preparative reversed-phase C18 column coupled to a high-pressure liquid chromatography (HPLC) system. The gradient used was from 20 to 80% buffer B in 60 min under 5 mL/min flow rate. The absorbance was monitored at 214 nm. (B) The protein content was evaluated by 12% SDS-PAGE electrophoresis in polyacrylamide gel (10 μg/well). Samples 1, 2, and 3 correspond to the fractions obtained by chromatography. MW corresponds to molecular mass markers, and the band circled in red refers to the TmC4-47.2.; Figure S2: (A) Second fractioning step for toxin purification. Fraction 2 (1 mg mL−1) obtained in the first chromatography was applied to a reversed-phase analytical C8 column coupled to a high-pressure liquid chromatography (HPLC) system. The gradient used was 20 to 80% of buffer B over 35 min under a flow rate of 1 mL/min. The absorbance was monitored at 214 nm. (B) The protein content of fractions 1, 2s, 2d, 3 and 4 was evaluated by 12% SDS-PAGE electrophoresis on polyacrylamide gel (10 μg/well). MW corresponds to molecular mass markers. The bands highlighted in red refer to TmC4-47.2 toxin; Figure S3: (A) Third fractionation for purification of *Thalassophryne maculosa* TmC4-47.2 toxin. Fractions 2s, 2d and 3 were mixed and 1 mg mL−<sup>1</sup> of the pool was applied to a reversed-phase analytical C8 column coupled to a high-pressure liquid

chromatography (HPLC) system. The gradient used was 20 to 80% buffer B over 35 min under 1 mL min−<sup>1</sup> flow rate. The absorbance was monitored at 214 nm. (B) The protein content of fractions 1, 2s and 2d was evaluated by 12% SDS-PAGE electrophoresis on polyacrylamide gel (10 μg/well). MW corresponds to molecular mass markers and the bands highlighted in red refer to TmC4-47.2 toxin.

**Author Contributions:** Methodology, validation, formal analysis, investigation, data curation, writing—original draft preparation, P.I.S.J., K.C., A.L.A.M., L.B.-L., G.R.D.; Conceptualization, resources, data curation, formal analysis, writing—review and editing, visualization, supervision, project administration, funding acquisition, M.L.-F., I.S.-R. and C.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the São Paulo Research Foundation—FAPESP (#2013/07467-1) and in part by the Coordination for the Improvement of Higher Education Personnel—CAPES (#001) and National Council for Scientific and Technological Development—CNPq (#305414/2019-4).

**Institutional Review Board Statement:** The study was conducted according to the animal welfare standards, and all necessary permits for the capture of *T. maculosa* and to collect the venom were obtained from the Escuela de Ciencias Aplicadas del Mar, Núcleo Nueva Esparta, Universidad de Oriente, Isla Margarita; Venezuela (Permit Number #30/07). Additional experiments were carried out under the National Council for Animal Experiment Control (CONCEA) and approved by the Butantan Institute's Animal Use Ethics Commission (CEUAIB #275/06).

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We thank the São Paulo Research Foundation—FAPESP, CAPES, and CNPq for the financial support. Additional thanks to Douglas Boletini-Santos for the assistance in the data collection and to the Butantan Institute Immunoregulation Unit staff. We also thank the anonymous reviewers for their careful reading and insightful comments and suggestions.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

### **References**

