Fig 2: Wnt3a proteins were recovered into the exosome pool, P100 pellet, prepared from conditioned medium at the apical or basolateral side of MDCK cells.(a) Western blot analysis to examine relative amounts of exosome marker proteins, CD63, CD81, Tsg101, and Hsp70, recovered in P100 pellets of apical or basolateral medium of Wnt3a-expressing MDCK (MDCK-3a) cells. Equal amounts of P100 samples prepared from apical or basolateral medium were subjected to Western blotting. As standards, small amounts of cell lysates were also loaded. (b) The amounts of Wnt3a in S10 sup and P100 pellet from apical or basolateral medium of MDCK-3a cells, as well as the amount in the cell lysate, were analyzed by Western blotting. To compare the amounts of Wnt3a proteins between apical and basolateral media, an equal volume of S10 (10 µl each for apical or basolateral S10 pool) or equal amount of P100 (1/40 of P100 pellets prepared from 400 ml of apical or basolateral media) samples was loaded. In all MDCK cultures in this study, the same volume of medium was added to both apical and basolateral chambers. (c–f) Western blotting analysis for detection of activity and amount of Wnt proteins recovered in S10 sup, S100 sup or P100 pellet prepared from either the apical (c) or basolateral side (e) of MDCK cells. For monitoring of the Wnt3a activity, the amount of stabilized ß-catenin induced by the addition of S10 sup, S100 sup or P100 pellet was quantified in L cells, in which ß-catenin is undetectable without the addition of Wnt proteins (control; c,e). In parallel, the amount of Wnt3a used in this activity assay was analyzed (c,e). The amounts of ß-catenin and Wnt3a in S10, S100 and P100, shown in (c) or (e), were quantified using Image J software. The ratios of ß-catenin to Wnt3a level are indicated in numerical values relative to that in S10, which was set as 100 (d,f). The results shown are the mean ± S.D. from 3 independent experiments. Full-length blots are presented in Supplemental Fig. S4.

Fig 3: Changes in (A) the relative metallothionein-10 mRNA levels [Type III (stronger) response] and in (B) the relative Hsp70 mRNA levels [Type III (stronger) response], and Hsp72 and 73 levels [Type II & III (faster and stronger) responses] in the mantle of heat hardened (H) and non-hardened (C) Mytilus galloprovincialis when exposed to 24 °C (a,d), 26 °C (b,e) and 28 °C (c,f) compared to control at 18 °C. Representative blots are shown. Blots were quantified using scanning densitometry. Full-length blots/gels are presented in Supplementary Fig. 2. Values are means ± SD, n = 8 preparations from different animals. *p < 0.05 compared to day 0, +p < 0.05 compared to non-hardened (C) mussels.

Fig 4: Double immunofluorescence with the two anti-HSP70 antibodies. Purified human spermatozoa were incubated in a capacitating medium for 4 h. They were then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton-X-100 and stained successively with polyclonal (Proteintech, 10995-1-AP, green labelling) and monoclonal (Sigma, H5147, red labelling) anti-HSP70 antibodies. Scale bar: 10 μm. Images are maximum-intensity projections (MaxIP) obtained from z stack images using Nikon NIS Elements software. A: acrosome, ES: equatorial segment, MP: mid-piece, N: neck, and T: tail.

Fig 5: (A) Representative pictures for high and low prelamin A immunostaining in locally advanced cervical cancer (LACC) patients (magnification 40x). (B) Representative western blot analysis of prelamin A expressions from patients who completely responded to treatment (pR0) and patients with macroscopic residual tumor (pR2) after chemoradiotherapy. #No.= patient code. (C) Densitometric analysis of normalized prelamin A levels respect to HSP70; columns represent the mean ± SEM for each group (n=10 for both groups). Difference was statistically significant between tumors from pR0 and pR2 groups (p < 0.05). (D) The relative mRNA expression of LMNA was evaluated by RT-PCR, utilizing specific set of primers. All samples were normalized to the housekeeping gene, B2M. Results are presented as the mean ± SEM of log2 fold change representative of mRNA expression levels relative to the whole population of samples (n=10 for both groups). (E) Representative pictures for low and high RAD51 immunostaining in LACC patients (magnification 40x). (F) Representative western blot analysis of RAD51 expressions from patients who completely responded to treatment (pR0) and patients with macroscopic residual tumor (pR2) after chemoradiotherapy. #No.= patient code. (G) Densitometric analysis of normalized RAD51 levels respect to HSP70; columns represent the mean ± SEM for each group (n=10 for both groups). Difference was statistically significant between tumors from pR0 and pR2 groups (p < 0.05). (H) The relative mRNA expression of RAD51 was evaluated by RT-PCR, utilizing specific set of primers. All samples were normalized to the housekeeping gene, B2M. Results are presented as the mean ± SEM of log2 fold change representative of mRNA expression levels relative to the whole population of samples (n=10 for both groups). (I) Kaplan–Meier survival curve for the probability of disease-free survival (left) and overall survival (right) according to expression of RAD51 in LACC patients. High expression of RAD51 was significantly associated with disease-free survival and overall survival disadvantage (p = 0.011 and p = 0.05, respectively).