Fig 2: PRG5 stimulates formation of dendritic protrusions, independent of neuronal activity. (A) Representative western blot after subcellular fractionation of adult mouse cerebral cortex, probed with PRG5, with PSD95 as a postsynaptic density marker, HSP60 as a mitochondrial marker, synaptophysin as a synaptic marker, and MBP as a myelin marker. PRG5 was detected in myelin-, ER-, Golgi-enriched, and in synaptosomal fractions, predominantly around 150 kDa. The 37 kDa PRG5 band was mainly detected in the myelin-enriched and the crude synaptic plasma-membrane fractions. No PRG5 signal was found in the postsynaptic density fraction. A representative blot of three technical repeats for each of three tissue preparations is shown. MW, molecular weight. (B) Representative volume-rendered 3D projections of z-stack images of murine primary neurons cultured 15 days in vitro and stained for PRG5 (green), homer1 (magenta), and bassoon (top, blue) or MAP2 (bottom, blue). Stainings were analyzed for three independent neuron preparations. PRG5 protein was not localized to pre- and postsynaptic transmission sites, as shown by a lack of signal overlap to homer1 or bassoon proteins (top, white arrow heads). PRG5 was localized at the bases of postsynaptic spines, between postsynaptic density protein homer1 and dendritic-cytoskeletal protein MAP2 (bottom, white arrow heads). Scale bars = 1 μm. (C) Quantification of protrusions of mature primary neurons transfected either with CFP-MEM (control) or PRG5-eGFP with (+) and without (–) activity inhibition (1 μM TTX, 50 μM D-APV and 10 μM nifedipine). PRG5- overexpressing neurons showed elevated number of overall protrusions and of homer-positive protrusions compared to control cells after inhibition of neuronal activity. Number of protrusions was normalized to control (CFP-MEM) without activity inhibition (100%) and are shown as mean in% + SEM. Counted numbers and n-values are listed in the table below graphs. Statistical data is shown in Supplementary Table 2.

Fig 3: Changes in abundance of selected HSPs in mitochondrial and cytosolic fractions following sub-lethal and PCD-inducing heat treatment. (A) Arabidopsis cell suspension culture (ecotype Col-0) was subjected to 10 min of control (25°C), sublethal (33°C), or PCD-inducing (54°C) temperature. Bars represent mean of three experiments ± SD for rates of PCD, necrosis, and viability 24 h following heat treatment. (B) For WB, 10 µg of proteins from cytosolic and mitochondrial fractions, isolated directly after heat treatment, were loaded per lane. Densitometry analysis was performed using GelAnalyzer. Bars represent mean signal intensity (± SD) relative to signal of mitochondrial control fraction (IDH2, HSP60, and mtHSP70) or control cytosolic fraction (ctHSP70). Means labeled with different letters are statistically different at 0.05 level (ANOVA, followed by Tukey HSD post-hoc test). AB, antibody; Sub, sublethal; Crtl, control. (C) Percentage of mitochondrial proteins released after PCD-inducing HS (54°C, 10 min). Bars represent mean of three experiments ± SEM, *p < 0.01 (paired Student’s t-test).