Cell culture, transfection, confocal microscopy, and image analysis

To determine whether the mutations result in HSPA1A misfolding or alter its intracellular localization we transfected mutated and WT GFP-tagged HSPA1A into human cells and visualized the protein using confocal microscopy. The rational of these experiments relies on the notion that if a mutation drastically alters protein structure, this may result in misfolding and improper intracellular localization.

Our focus was to determine the effect of the mutation on the protein and not to study the behavior of cells carrying these mutated HspA1A variants under stress. For this purpose, we used HEK293 cells (which were purchased from ATCC; ATCC^® CRL-1573^™). The cells were maintained in a humidified 5% CO₂ atmosphere at 37 °C in complete medium consisting of DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin. A day before transfection cells were split into 24-well plates at 2.0 × 10³ cells/well containing poly-D-lysine treated coverslips. After 18 hours cells were transiently transfected with the WT or mutant HSPA1A construct using the PolyJet In Vitro DNA Transfection Reagent (SignaGen) as per the manufacturer’s instructions. Transfection was allowed to continue for 18 hours, and then transfection media was removed and replaced with fresh complete media. At that time cells were either maintained at 37 °C or were placed in a water-bath at 42 °C for 60 minutes. After stress, the nucleus was stained using DAPI (present in the Fluoromount-G mounting media; Southern Biotech), the mitochondria using MitoTracker Red (100 nM; Life Sciences), and the plasma membrane using wheat germ agglutinin-AF555 (500 ng/mL; Life Sciences). The lysosomes were visualized by co-transfecting the HSPA1A variant with the lysosome-associated membrane protein 1 (LAMP1)-mRFPC1 construct [a gift from Walther Mothes (Addgene plasmid # 1817)]. After staining, cells were fixed using freshly prepared solution of 4% paraformaldehyde in complete growth medium. Cells were visualized using a Leica DM IRE2 confocal microscope equipped with a 63 × 1.4 oil objective. Image and co-localization analyses were performed for 15 cells from three independent experiments. Images were analyzed in ImageJ⁵⁹ using the corrected total cell fluorescence (CTCF) method⁶⁰ as a ratio between the total GFP-HspA1A fluorescence of a particular compartment and the rest of the cell. Statistical significance was determined by one way Anova with post-hoc Tukey HSD (honest significant difference) and Holm multiple comparison tests. A p-value < 0.05 from both post-hoc tests was considered statistically significant.