Fig 2: Hydrophobic Shielding Reduces the Ability of aS Oligomers to Induce Membrane Disruption and ROS Production(A and B) BisANS inhibits the binding of CLU (A) and a2M (B) to aS oligomers. The chaperones (present at 10 µg · mL-1) were incubated in an ELISA plate pre-coated with aggregated aS, and the amount of bound chaperone was then measured by ELISA. Neither chaperone bound to monomeric aS or the BSA blocker (Figure S3). Data shown are means ± SD of three independent experiments.(C) aS oligomers were pre-incubated with CLU, a2M, or BSA and then added to surface-tethered lipid vesicles filled with the Ca2+-sensitive fluorophore Cal-520. The extent of membrane permeabilization was quantified as a percentage of maximum fluorescence produced after incubation with the Ca2+ ionophore ionomycin. Example images of vesicles after the addition of the indicated sample are shown. Scale bar (bottom right), 2 µm.(D) Quantification of the data shown in (C). a2M or CLU (0.05–50 nM) was incubated with aggregated aS. The extent of membrane permeabilization decreased with increasing chaperone concentration. Data shown are means ± SD of 9 fields of view (at least 800 vesicles) and are representative of two independent experiments.(E) Aggregated aS preincubated with CLU, a2M, or BSA (each present at a 1:10 substoichiometric ratio) was added to Neuro-2a (N2a) cells. The rate of ROS production before and after the sample addition was quantified by measuring the oxidation of DHE to ethidium by epifluorescence microscopy. The change in the rate of ROS generation due to the addition of a sample was calculated by subtracting the gradient of the pre-addition line from the gradient of the post-addition line. Example rates of ROS production in a single cell under the indicated conditions are shown.(F) Quantification of the data shown in (E). The change in the rate of ROS production produced by each sample relative to the buffer-only sample is indicated. The values are means ± SD of approximately 50 cells across three replicate treatments. **p < 0.01, analyzed by one-way ANOVA with a Bonferroni post-test.

Fig 3: The Ratio of CLU:aS Decreases in Larger OligomersaSA90C-AF488 (70 µM) and CLU-AF647 (0.7 µM) were co-incubated in PBS (pH 7.4) at 37°C, with shaking at 200 rpm. The formation of aS-CLU complexes was quantified by single-molecule TCCD.(A and B) Contour plots of the apparent number of aS monomers constituting an oligomer as a function of the ZCLU/aS value for samples taken from the aggregation reaction after 6 hr (A) and 48 hr (B), respectively. ZCLU/aS represents the logarithm of the apparent ratio of CLU to aS in each oligomer. The data shown are representative of three separate experiments. The numbers in the inserts indicate the number of complexes represented in the plot.(C and D) Frequency histograms of the number of oligomers at different ZCLU/aS values (for the data shown in A and B, respectively). The dotted lines each indicate a specific CLU: aSA90C stoichiometry (as shown on the upper x axis). The data are representative of three independent experiments.

Fig 4: Expression of sCLU isoform from OP patients and CTR subjects. (A) CLU expression level was analyzed in osteoblasts from 7 OP patients and 7 CTR subjects. OP patients show an increased expression level of CLU with respect to CTR (** p < 0.01). (B) CLU expression level was analyzed in PBMCs from 30 OP and 30 CTR. OP samples show an increased expression level of CLU with respect to CTR (* p < 0.05). GAPDH mRNA level was used to normalize the relative amount of CLU and relative expression values are expressed as 2-??CT.

Fig 5: Effects of combined CSE treatment and CLU silencing on proliferation and activation of human lung fibroblasts. (A) Effects of CSE treatment and CLU silencing on cell proliferation. (B) Effects of CSE treatment and CLU silencing on the expression of apoptosis-related proteins Bax, Bcl-2, caspase3, p53, and p21. (C) Effects of CSE treatment and CLU silencing on the expression of inflammatory cytokines IL-6, IL-8, and TNF-a. (D) Effects of CSE treatment and CLU silencing on a-SMA and FN expression. (E) Effects of CSE treatment and CLU silencing on the expression of collagens I and III. (F) Effects of CSE treatment and CLU silencing on MMP-2 and MMP-9 expression. Data are presented as means ± SDs of three independent experiments, with GAPDH as an internal control. a-SMA: a-Smooth muscle actin; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma-2; CLU: Clusterin; CSE: Cigarette smoke extract; FN: Fibronectin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IL: Interleukin; MMP: Matrix metalloproteinase; NC: Negative control; ns: Not significant; SD: Standard deviation; TNF-a: Tumor necrosis factor a. * P < 0.001, † P < 0.01, ‡ P < 0.05.