Extended Data Fig. 3: RNA characterization of aggresomes.
From: Aggresomes protect mRNA under stress in Escherichia coli
a. Distribution of RNA lengths in aggresomes versus cytoplasm, analysed using transcriptome sequencing data aligned to operonic mRNA references. b. Aggresome-to-cytoplasm RNA ratio as a function of transcript length (nucleotides), derived from transcriptome sequencing data referenced against operonic mRNAs. c, d. Aggresome morphology by transmission electron microscopy (TEM), Scale bar: 500 nm. c. Representative TEM images post-arsenite treatment. d. Aggresome area quantification from TEM data (n = 20 cells per condition, mean ± SE). e–j. Analysis of aggresome compaction via SIM: e, g, i. SIM images of cells expressing nuoJ-8pepper (e), nlpE-8pepper (g), or mukF-8pepper (i) after indicated arsenite (NaAsO2) treatment durations. HBC530 dye was used for RNA visualization, Scale bar: 500 nm. f, h, j. Aggresome area quantification from e, g, and i, respectively (n = 10 cells per condition). k. Protein mass per aggresome after arsenite treatment: Protein total: Bulk aggresome protein (Qubit fluorometry). Naggresome=Aggresome count (FACS). Protein per aggresome = Protein total / Naggresom (n = 3 independent biological replicates, mean ± SE). l. Workflow for in vitro single-molecule mRNA detection: Left: Pepper RNA (stem: orange; aptamer: blue) immobilized on a passivated coverslip and incubated with HBC530 dye. Middle: Slimfield microscopy localizes dye-bound complexes as diffraction-limited foci (ms timescale). Right: Custom single-particle tracking software determines centroid positions (~40 nm precision) and quantifies focus brightness (modal intensity: ~90 counts; background-subtracted), Scale bars: 1 μm. Two-sided unpaired Student’s t-test used in comparison; error bars indicate SE.
