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Additional file 14. A table with description of strains and plasmids generated and used in this study and graphical representation of over-expressed operons/genes

Human Stefin B Role in Cell's Response to Misfolded Proteins and Autophagy

<div><p>Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in <i>Saccharomyces cerevisiae</i> expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.</p></div

Yeast <i>S. cerevisiae</i> genes in positive genetic interaction with stefin B gene (cystatin B gene - <i>CSTB</i>).

<p>Functions of the encoded proteins according to SGD (<a href="http://www.yeastgenome.org/" target="_blank">http://www.yeastgenome.org/</a>) are shown. R denotes relative growth rate compared to the reference strain (average of two biological replicates). Genes that have a function in vesicular transport are stressed in <b>bold</b>. Two proteins whose function is related to autophagy are also given in <b>bold</b>.</p

Yeast <i>S. cerevisiae</i> genes in negative genetic interaction with stefin B gene (cystatin B gene - <i>CSTB</i>).

<p>Functions of the encoded proteins according to SGD (<a href="http://www.yeastgenome.org/" target="_blank">http://www.yeastgenome.org/</a>) are shown. R denotes relative growth rate compared to the reference strain (average of two biological replicates). A gene that has a function in polyamine transport is written in <b>bold</b> as well as a gene involved in inositol phosphates pathways, up-stream of autophagy.</p

Fluorescence images of stefin B deficient (KO) and wild type (wt) astrocytes isolated from FVB mice stained with Proteostat and DAPI.

<p>KO cells were incubated with 30 µM stefin B proteins or 20 nM rapamycin for 24 hours. A: KO astrocytes control, B: KO astrocytes + monomers, C: KO astrocytes + dimers, D: KO astrocytes + tetramers, E: KO astrocytes + oligomers, F: wt astrocytes, G: KO astrocytes + rapamycin. Scale bar: 10 µm (except G: 20 µm).</p

Bimolecular fluorescence microscopy - dimerization of stefin B in HEK293 cells.

<p>Cells were transfected with different combinations of plasmids to follow dimerization of over expressed proteins. For each protein a yellow fluorescent N-terminal (NYFP) or C-terminal half (CYFP), respectively, was attached to the C-terminus of the desired protein. A: control: wild type (WT)-NYFP + SUMO-CYFP; B: WT-NYFP + WT-CYFP; C: G4R-NYFP + G4R-CYFP; D: G4R-NYFP + WT-CYFP. SUMO protein was selected as the control protein due to its high solubility and abundance. Scale bar: 10 µm.</p

Western blot analysis (A) of the protein expression of p62 and LC3-I and LC3-II in primary FVB +/+ (wild type) and StB −/− (knock out) astrocytes.

<p>(B) Western blot analysis of the protein expression of LC3-I and LC3-II in primary stefin B KO astrocytes after incubation with cathepsin inhibitor. 20 nM rapamycin and 5 µM E-64d were added to the medium for 24 hours. Cells were incubated with 80 nM bafilomycin for 30 minutes.</p

Shift in Bodipy 581/591 C₁₁ fluorescence measured by flow cytometer.

<p>Bodipy 581/591 C₁₁ was excited using a 488 nm Ar-laser and detected on a FL1 photodetector (515∼545 nm) on flow cytometer. Untreated FVB +/+ wild type (wt) astrocytes had lower lipid peroxidation signal compared to StB −/− knock out (KO) cells. KO cells were more sensitive to 1 mM H₂O₂ (30′) treatment. The representative results of one experiment (out of three) are shown.</p

Western blot analysis of the protein expression of LC3-I and LC3-II in primary astrocytes after the addition of stefin B to the medium.

<p>Stefin B deficient (KO) astrocytes were incubated for 24 hours with different forms of the wild type (wt) stefin B (A) and G4R mutant (C) protein as stated on the image. Below each immunoblot a graphic representation of densitometric analysis of three (wt stefin B) (B) or two (G4R mutant) (D) independent experiments is shown. For positive control, 20 nM rapamycin was added for 24 hours. Cells were incubated with 80 nM bafilomycin for 30 minutes.</p

MOESM4 of Integrated omics approaches provide strategies for rapid erythromycin yield increase in Saccharopolyspora erythraea

Additional file 4. A table for validation of microarray data by qPCR