Fig 1: Experimental setup and results of colocalization analyses of septin-3 (SEPT3) due treatments. A Embryos were excised from three different dams to create biological triplicates of cultures. Embryonic cortices were isolated and homogenized to prepare cortical primary neuronal cell cultures. Two wells per culture were exposed to treatments (either AUTEN-67 or CCCP), two wells per culture were used as control. Cells were immunostained against SEPT3, LC3B and PINK1. Three images were captured and analyzed per well for every treatment/control condition. B–D Percentage comparison of colocalizing signals between treated and control cells. Means are marked with horizontal lines with values; boxes represent standard error of the mean (SEM). Flags indicate standard deviation (SD). In the figure legend, results are presented as mean ± SEM; SD. The p-values are of independent two-tailed Student’s t-test. B LC3B colocalizing SEPT3 in AUTEN-67 treatments, 13.01% ± 1.21%; 5.14%. Controls: 9.26% ± 1.1%; 4.68%. Significance: p = 0.0283. C LC3B colocalizing SEPT3, CCCP treatments: 10.49% ± 1.53%; 6.50%. Controls: 4.01% ± 1.06%; 4.38%. Significance: p = 0.0016. D PINK1 colocalizing SEPT3, CCCP treatment: 8.07% ± 1.52%; 6.46%. Controls: 8.65% ± 1.74%; 7.19%. Significance: p = 0.80
Fig 2: Protein–protein interaction measurements of septin-3 (SEPT3) and Atg8 homologs. A Microscale thermophoresis dose–response curves of LC3B-SEPT3 (yellow) and GABARAPL2-SEPT3 interactions (blue). X axis represents SEPT3 concentrations on a log scale, while Y axis reflects MST dose–response, normalized to baseline (Fnorm). Corresponding KD values and KD confidence are displayed. B Spectral shift fluorescence response of LC3B-SEPT3 (yellow) and GABARAPL2-SEPT3 (blue). C Fluorescence polarization direct titration curves of LC3B-SEPT3 (marked yellow) and GABARAPL2-SEPT3 (marked blue) interactions. X axis represents SEPT3 concentrations on a linear scale, while Y axis reflects polarization
Fig 3: Confocal microscopy images of primary neuronal cells. A Representative image of enriched LC3B positive particles in the cell body of an AUTEN-67 treated primary neuron indicate enhanced autophagy compared to control. B Overlapping spots of fluorescent signals along the axons (left) and in the cell body (right). C Enlarged and resolved images of overlapping signals from B, with observable LC3B and septin-3 (SEPT3) colocalization, where SEPT3’s colocalization with PINK1 is minimal. However, more complex colocalization patterns of LC3B–PINK1–SEPT3 can be observed from other parts of the sample (D)
Fig 4: Sequential and structural location of LIR motifs in septin-3. A Positions of five ELM predicted LIR motifs on the canonical human SEPT3 A chain, with secondary structure display. The most likely functional LIR4 is highlighted with light blue, while LIR1, 2, 3, and 5 are shown in dark blue. LIR4-overlapping SUMO interacting motif (SIM) is marked with purple. B factor analysis scale of residues 63–329 reveals the highly mobile loop regions of the molecule. B ELM Predicted LIR motif sequences of SEPT3 by the canonical LIR motif θ0-X1-X2-Γ3, where θ marks an aromatic, Γ an aliphatic, and X an arbitrary residue. Θ and Γ residues are shown in bold, other key residues determining interactions or functionality (described in the text) are shown with grey background. C, D Molecular dynamics (MD) simulations of septin-3 structure. C Septin-3 structural mobility is visualized by B-factor representation. LIR4 motif is located at a highly flexible, exposed region of the molecule (light blue). D Superposition of representative structure ensemble from MD trajectory. LIR motifs are colored as in (A)
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