Journal: bioRxiv

Article Title: Chemical factors induce aggregative multicellularity in a close unicellular relative of animals

doi: 10.1101/2022.12.01.517680

Figure Lengend Snippet: (A) 5% (v/v) of proteinase-treated >30 kDa FBS fraction shows reduced aggregation activity compared to 5% (v/v) of untreated >30 kDa fraction in combination with 0.2 mM CaCl 2 . A 10% (v/v) treatment of 1X PBS was used as negative control. Data are represented as boxplots, showing the median aggregate area (thick black bar) and interquartile ranges from three independent biological replicates. Figure related to - . (B) ApoB100 in combination with 0.3 mM CaCl 2 does not fully recapitulate the aggregation activity of 10% (v/v) FBS. Data are represented as boxplots, showing the median aggregate area (thick black bar) and interquartile ranges from two independent biological replicates in agitation conditions ( assay) . A 10% (v/v) of FBS was used as a positive control and a 10% (v/v) of 1X PBS was used as a negative control. Figure related to and Table S1 . (C) Dose-response of whole LDLs (plus 0.2 mM CaCl 2 ) in the absence of agitation conditions ( assay) . Data are represented as the average aggregate area ± s.e.m. from three independent biological replicates. (D) Dose-responses of whole FBS and LDL-deficient FBS (both in the presence of 0.2 mM CaCl 2 ) in the absence of agitation ( assay) . LDL-deficient FBS has reduced activity, inducing weak aggregation at 30% (v/v) concentrations, whereas whole FBS induces aggregation at ∼1% (v/v). Data are represented as the average aggregate area ± s.e.m. from three independent biological replicates. Figure related to . (E) Dose-response of protein-free LDL particles (plus 0.2 mM CaCl 2 ) in the absence of agitation conditions ( assay) . Data are represented as the average aggregate area ± s.e.m. from three independent biological replicates.

Article Snippet: A 5% (v/v) of washed >30 kDa FBS fraction was premixed with a 1:3 serial dilution of CaCl 2 (#C5080, Sigma-Aldrich) diluted in 1X PBS (concentration range from 0.5 to 0 mM) and later added to each well.

Techniques: Activity Assay, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Chemical factors induce aggregative multicellularity in a close unicellular relative of animals

doi: 10.1101/2022.12.01.517680

Figure Lengend Snippet: The aggregation activity of >30 kDa FBS fraction components was tested by treating Capsaspora cells in FBS-free media with 0.2 mM CaCl 2 (in all conditions) and either one of the following components: (A) nothing else added (negative control); (B) complete >30 kDa FBS components (positive control); (C) re-dissolved 20% ammonium sulfate precipitate; (D) re-dissolved 40% ammonium sulfate precipitate; (E) re-dissolved 65% ammonium sulfate precipitate; (F) re-dissolved 80% ammonium sulfate precipitate; and (G) washed supernatant remaining from 80% ammonium sulfate precipitation. Scale bar represents 100 μm.

Article Snippet: A 5% (v/v) of washed >30 kDa FBS fraction was premixed with a 1:3 serial dilution of CaCl 2 (#C5080, Sigma-Aldrich) diluted in 1X PBS (concentration range from 0.5 to 0 mM) and later added to each well.

Techniques: Activity Assay, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Chemical factors induce aggregative multicellularity in a close unicellular relative of animals

doi: 10.1101/2022.12.01.517680

Figure Lengend Snippet: (A-B) Coomassie-stained gels of active fractions “1” (shown in A) and “2” and “3” (shown in B) from size exclusion chromatography, related to supplementary . Right images in A-B show boxes around the bands (#1-7) that were excised and subjected to in-gel trypsin digestion and LC-MS/MS. The primary protein of each band was Apolipoprotein B100 (ApoB100, see Table S1 ). (C) ApoB100 in combination with 0.3 mM CaCl 2 does not recapitulate the aggregation activity of 10% (v/v) FBS. At 120 µg/mL, ApoB100 appears toxic. A 10% (v/v) of 1X PBS was used as a negative control. Scale bar represents 100 μm. Microscopy images representative of each test condition in .

Article Snippet: A 5% (v/v) of washed >30 kDa FBS fraction was premixed with a 1:3 serial dilution of CaCl 2 (#C5080, Sigma-Aldrich) diluted in 1X PBS (concentration range from 0.5 to 0 mM) and later added to each well.

Techniques: Staining, Size-exclusion Chromatography, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Negative Control, Microscopy