s2 cell pellets  (Thermo Fisher)

Journal: Life Science Alliance

Article Title: Selective autophagy fine-tunes Stat92E activity by degrading Su(var)2-10/PIAS in Drosophila glia

doi: 10.26508/lsa.202503375

Figure Lengend Snippet: (A, B) Fluorescent microscopic images of uninjured and injured wing nerves 3 dpi. Single longitudinal optical slices are shown in each case with a single transversal slice for the injured specimens. repo-Gal4 –driven myr::mtdTomato colocalizes with the signal from the 10xStat92E enhancer-GFP and the TRE-EGFP reporters in glia. Please note that the elongated fibers that are outlined by reporter expression could potentially correspond to either glia or axons. Glia, in the wing nerve and generally in the peripheral nervous system, especially wrapping glia, have a filament-like appearance that can resemble axons . Arrowheads denote glial nuclei. Scale bar: 5 μm. (C, D) Fluorescent microscopic images of neuronal cell bodies of the wing nerve in injured anterior wing margins at 3 dpi. Arrows point to neuronal cell bodies, which occupy a stereotypical position at the base of the sensory bristles at the sensilla. Single optical slices are shown in each case. Signal of the nSyb-LexA –driven mtdTomato + cell bodies does not colocalize with the signal from the 10xStat92E enhancer-GFP and the TRE-EGFP reporters in neuronal cell bodies. Neuronal expression of TRE-EGFP and 10xStat92E enhancer-GFP therefore can be excluded because these reporters do not outline the wing vein neuronal somata. Scale bar: 10 μm.

Article Snippet: S2 cell pellets were lysed on ice for 30 min in three times packed volume RIPA buffer (50 mM Tris, pH = 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, Pierce Protease Inhibitor Tablet [A32963; Thermo Fisher Scientific], Halt Protease and Phosphatase Inhibitor Cocktail [78442; Thermo Fisher Scientific]) and cleared by centrifugation at 4°C.

Techniques: Expressing

Journal: Life Science Alliance

Article Title: Selective autophagy fine-tunes Stat92E activity by degrading Su(var)2-10/PIAS in Drosophila glia

doi: 10.26508/lsa.202503375

Figure Lengend Snippet: (A) Single-slice images of wing nerves showing 10xStat92E enhancer-GFP signal upon glial RNAi. RNAi expression was induced by RU486 for 5–7 d after eclosion before injury and lasted for the duration of the experiment. Atg16 RNAi effect is not abrogated by simultaneously introducing a second UAS construct ( UAS-mtdTomato ) arguing against Gal4 unavailability on Atg16 RNAi and resulting derepression of 10xStat92E enhancer-GFP in . The boxed area indicates the position of wing nerve glia adjacent to epithelial GFP signal. Scale bar: 5 µ m. (B) Quantitative analysis of 10xStat92E enhancer-GFP signal shown in (A). Truncated violin plots are shown with median and quartiles. We used unpaired, two-tailed Mann–Whitney test for the analyses. ** P = 0.0033, ns = 0.4491. n = 9, 13, 11, 12. (C) Single optical slices showing Su(var)2-10 abundance in brain glia around the antennal lobe in glial luc and Atg1 KD, without injury and 1 d after antennal ablation, respectively. Su(var)2-10 is predominantly nuclear. Glial nuclei labeled by repo-Gal4>NLS-GFP are outlined. Note the accumulation of Su(var)2-10 in glial nuclei, and that a fraction of NLS-GFP is also present in the cytoplasm. Scale bar: 5 μm. (D, E) Quantitative analysis of Su(var)2-10 nuclear intensity normalized to background signal in single optical slices shown in (C). An unpaired, two-tailed Mann–Whitney test and t test were used for statistics for uninjured and injured pairwise comparisons, respectively. **** P < 0.0001, ns, not significant. n (D) = 59, 58. n (E) = 80, 110. (F) S2 cell extracts transiently expressing Su(var)2-10-EGFP were immunoblotted for GFP (GFP, magenta) and Su(var)2-10 (Su(v), green) simultaneously. Individual channels and the merged image (Mer) are shown. The signals of the reactive bands completely colocalize indicating recognition of Su(var)2-10 by the Su(var)2-10 antibody. Control extract (C) not expressing Su(var)2-10::GFP is displayed for comparison on the left. Anti-α-tubulin serves as a loading control. (G) Homozygous Su(var)2-10 MI03442 loss-of-function allele and control (C) w 1118 L3-stage larva extracts were immunoblotted for Su(var)2-10 and β-actin. Note the reduced Su(var)2-10 isoform band intensities in the mutant in the >∼45 kD size range. Asterisks indicate bands that are not affected by the MiMIC insertion, most likely nonspecific bands in larva.

Article Snippet: S2 cell pellets were lysed on ice for 30 min in three times packed volume RIPA buffer (50 mM Tris, pH = 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, Pierce Protease Inhibitor Tablet [A32963; Thermo Fisher Scientific], Halt Protease and Phosphatase Inhibitor Cocktail [78442; Thermo Fisher Scientific]) and cleared by centrifugation at 4°C.

Techniques: Expressing, Construct, Two Tailed Test, MANN-WHITNEY, Labeling, Control, Comparison, Mutagenesis

Journal: Life Science Alliance

Article Title: Selective autophagy fine-tunes Stat92E activity by degrading Su(var)2-10/PIAS in Drosophila glia

doi: 10.26508/lsa.202503375

Figure Lengend Snippet: (A) Single optical brain slice around the dorsal antennal lobe area is shown. Magnified images of one typical glial nucleus, denoted by an orange arrowhead, are shown below. Arrowheads point to glial nuclei, whereas arrows denote the exact location of Su(var)2-10-SUMO-Atg8a colocalization in the magnified image. Nuclei are stained with DAPI; magenta indicates native mApple::Atg8a signal from expression in glia by repo-Gal4 , combined with anti-Su(var)2-10 (green) and anti-SUMO (yellow) immunostaining. Scale bar for brain images: 10 μm; scale bar for nucleus images: 2 μm. (B) Confocal single-slice multichannel images show Su(var)2-10-Atg8a colocalization in the cytoplasm of glial cells. Magnified images of the areas outlined by the rectangles can be seen below. Arrows point to the exact location of the colocalization. Nuclei are stained with DAPI; mApple::Atg8a expression is driven in glia by repo-Gal4 , and its signal was amplified by anti-mCherry staining, jointly with anti-Su(var)2-10 immunostaining. Scale bar for brain images: 10 μm; scale bar for nucleus images: 2 μm. (C) S2 cells transiently expressing WT and autoSUMOylation-defective CTD2 mutant Su(var)2-10::EGFP were treated with bafilomycin A1 (BafA1) or the solvent DMSO for 4 h, and total Su(var)2-10 levels were assessed by anti-GFP Western blot. α-Tubulin serves as a loading control. (D) Quantification of Su(var)2-10 abundance normalized to α-tubulin as in (C). Shown are the mean and SD; one-way ANOVA with the Holm–Šídák test for multiple comparisons correction was used for statistics. n = 4, 3, 4, 4. (E) Co-immunoprecipitation of Su(var)2-10 with mCherry::Atg8a. Ubiquitous daughterless ( da )- Gal4 –driven myr::tdTomato (Tomato) or mCherry::Atg8a (Ch::Atg8a) whole adult fly extracts were precipitated with RFP-Trap beads. myr::tdTomato serves as a negative control. Eluates are shown on the right, and crude extract inputs (10 μg) on the left. The two major bands in mCherry::Atg8a correspond to the lipidated and unlipidated forms. Western blots were probed by anti-mCherry and anti-Su(var)2-10. Source data are available for this figure.

Article Snippet: S2 cell pellets were lysed on ice for 30 min in three times packed volume RIPA buffer (50 mM Tris, pH = 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, Pierce Protease Inhibitor Tablet [A32963; Thermo Fisher Scientific], Halt Protease and Phosphatase Inhibitor Cocktail [78442; Thermo Fisher Scientific]) and cleared by centrifugation at 4°C.

Techniques: Slice Preparation, Staining, Expressing, Immunostaining, Amplification, Mutagenesis, Solvent, Western Blot, Control, Immunoprecipitation, Negative Control