Fig 1: Hsp90 directly binds and is sufficient to align Collagen-1. (A). Collagen-1 gels were treated with purified FLAG- Hsp90α WT mixed in PBS and incubated for 5 days at 37 °C. LOXL2 was used as the positive control, and BSA was used as the negative control. SHG images were acquired spanning a 70 μm vertical distance (1 image/1 μm) starting at 200 μm from the base of the gel. The images shown in the figure are representative images of average projections of z-stacks of each condition. The white line in the images is a scale bar depicting a 25 μm distance. (B). Quantification of local variance of the Collagen-1 fibers analyzed from z-stack images obtained (one-way ANOVA; p-value < 0.0001). (C). Collagen-1 gels were treated with FLAG- Hsp90α WT and 7191 mixed in PBS and incubated for 5 days at 370. Controls were used as in Figure 3A. SHG images were acquired on day 5, spanning 70 μm with 1 image/1 μm. The figure shows representative average intensity projections of z-stacks of images captured for each condition. White lines shown in the images are scale bars representing 10 μm. (D). Quantification of local variance of the Collagen-1 fibers in z-stack images acquired (one-way ANOVA; p-value < 0.0001). (B,D). p value for pairwise comparison is represented as follows—<0.1234 as “NS”, <0.0021 as “**”, and <0.0001 as “****”. (E). Schematic of Collagen-1 binding assay. (F). A collagen-1 binding assay with recombinant FLAG-Hsp90α WT was performed. Linear regression of absorbance values with one site-specific binding is plotted, Kd = 1.9 nM. (G). Collagen-1 binding assay with FLAG-Hsp90α WT alone and FLAG-Hsp90α WT incubated with 10 nM of 7191. Linear regression with one site-specific binding—Kd = 14.74 nM (paired t-test; p-value = 0.0006). (F,G). p value for pairwise comparison is represented as follows—0.0002 as “***”.
Fig 2: Hsp90’s ATPase activity is not critical for binding and aligning Collagen-1; however, slowing ATPase activity increases Collagen-1 alignment. (A). Collagen-1 binding assay with FLAG-Hsp90α WT alone and combined with 100 μM ATPγS. Absorbance was measured at 607 nM and normalized to absorbance at FLAG- Hsp90α WT treatment at 0 nM. Linear regression of absorbance values with one site-specific binding is plotted (paired t-test; p-value < 0.0001). p value for pairwise comparison is represented as follows—<0.1234 as “NS”. (B). Collagen-1 gels were treated with FLAG- Hsp90α WT and 100 μM ATPγS mixed in PBS and incubated for 5 days at 37 °C. LOXL2 (positive) and BSA (negative) treatments were controls. SHG images were acquired on day 5, spanning 70 μm (1 image/1 μm) starting at 200 μm above the base of the gel. The images shown in the figure are representative images of average projections of z-stacks of each condition. The white line in the images depicts a 10 μm distance. (C). Quantification of local fiber variance of Collagen-1 gel images (one-way ANOVA; p < 0.0001). p value for pairwise comparison is represented as follows—<0.1234 as “NS”, <0.0332 as “*”, <0.0021 as “**”, and <0.0001 as “****”.
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