Fig 2: Downregulation of PrPC aggregates ß1-integrin and upregulates laminin and its receptor. (a) Silencing of PrPC in human TM cells followed by immunostaining with antibody specific for activated ß1-integrin shows clustering of activated ß1-integrin on the plasma membrane in the absence of PrPC (arrowheads) (panels 1–4). Cells transfected with PrP-siRNA and reacted with mouse IgG and respective secondary antibody do not show any reaction (panel 5). Scale bar: 25 µm. (b) Probing of TM cell lysates for PrPC shows the expected glycoforms in control cells transfected with scrambled siRNA, and minimal reaction in cells exposed to PrP-siRNA (lanes 1 & 2). Probing for laminin and laminin receptor (LR) shows significant upregulation in the absence of PrPC relative to control (lanes 1 & 2). (c) Quantification by densitometry after normalization with ß-actin shows 2-fold upregulation of laminin and LR due to downregulation of PrPC. Values are mean ± SEM of the indicated n. *p < 0.05. Full-length blots are included in the Supplementary Fig. S2.

Fig 3: Hypothetical representation of PrPC-mediated Endo-MT-like change in the TM. Physiological role of PrPC in the TM: (1) PrPC is expressed on the plasma membrane of TM cells. (2) PrPC maintains cell-ECM interactions by stabilizing ß1-integrin and other proteins5,7. Pathological implications: (1) Downregulation of PrPC induces (2) aggregation of ß1-integrin and (3) upregulation of fibronectin, collagen 1A, a-SMA, vimentin, and laminin, resulting in (4) increase in IOP and possibly POAG. AH: aqueous humor; PM: plasma membrane; Nu: nucleus.

Fig 4: Downregulation of PrPC upregulates vimentin and collagen 1A. (a) PrPC was silenced in human TM cells and lysates were processed as above. Probing for vimentin and collagen 1A shows significant upregulation in the absence of PrPC relative to controls (lanes 1 & 2). (b) Quantification of protein expression by densitometry after normalization with ß-actin shows 1.9-fold upregulation of vimentin and 2.2-fold upregulation of collagen 1A in the absence of PrPC. Values are mean ± SEM of the indicated n. *p < 0.05, **p < 0.01. Full-length blots (Supplementary Fig. S2). (c & d) Immunoreaction of fixed sections from the anterior segment of PrP+/+ and PrP-/- mice for vimentin and collagen 1A shows stronger reaction in the TM of PrP-/- relative to PrP+/+ samples (panel 1 vs. 2). No reaction was detected when serial sections were reacted with mouse or rabbit IgG followed by Alexa 546-conjugated secondary antibody (panels 5 & 6). Scale bar: 25 µm.

Fig 5: Distribution of PrPC in the human trabecular meshwork. (a) Immunoreaction of human TM section with PrP-specific antibody 3F4 followed by Alexa fluor 546-conjugated secondary antibody shows strong reactivity in all layers of the TM (panel 1). High magnification image demonstrates expression of PrPC on the plasma membrane of TM cells (panel 2). A serial section reacted with mouse IgG and Alexa fluor 546-conjugated secondary antibody shows no reaction (panel 3). H&E staining of a serial section confirms the TM region and Schlemm’s canal (SC) (panel 4). Scale bar: 25 µm. (b) Non-permeabilized primary human TM cells reacted with 3F4 followed by Alexa Fluor 488-conjugated secondary antibody show expression of PrPC on the plasma membrane (panel 1). No reaction is detected in control cells exposed to mouse IgG followed by the same secondary antibody (panel 2). Scale bar: 25 µm.