Dulbecco phosphate buffered saline (dpbs)

For the morphological observations, VSMCs were cultured on PLGA, GO-PLGA, RGD-PLGA and RGD-GO-PLGA mats for 3 days. The cells were then fixed in formalin solution (3.7% of formaldehyde in DPBS, Sigma-Aldrich Co.) for 10 min, and were immersed in 0.1% of Triton X-100 in DPBS (Sigma-Aldrich Co.) for 5 min. A 2% of bovine serum albumin (BSA, GenDEPOT, Barker, TX, USA) was added to block non-specific binding of antibodies for another 30 min, and the cells were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (at 1:40 in 1% of BSA solution in DPBS; Molecular Probes, Eugene, OR, USA) at room temperature for 20 min. The RGD-M13 phages functionalized in the RGD-PLGA and RGD-GO-PLGA mats were immunofluorescence stained using an α-M13 phage antibody (at 1:250 in 2% of BSA solution in DPBS; Sigma-Aldrich Co.) and a secondary fluorescein isothiocyanate (FITC)-labeled goat α-rabbit IgG (at 1:500 in 2% of BSA solution in DPBS; Abcam Inc., Cambridge, MA, USA) for 2 and 1 h, respectively. The nucleus was stained using 4’,6-diamidino-2-phenylindole (DAPI, 1 μM, Sigma-Aldrich Co.) solution in DPBS. All imaging was carried out using a custom-made two-photon laser fluorescence microscopy and immunofluorescence images analyzed with ImageJ 1.48v software (NIH, Bethesda, MD, USA) [35 (link), 36 (link)].

Western Blotting was carried out as previous described [24 (link)]. Briefly, cells were lysed in cold RIPA buffer (Life Technologies). The protein concentration was determined using the BCA Protein Assay Kit (Pierce Chemical, Rockford, IL, USA). Protein was separated with 10% SDS-PAGE, transferred to a PVDF membrane (Life Technologies), and then blocked in 5% nonfat dried milk (Yili, Beijing, China) in DPBS (Life Technologies) for 4 h. The PVDF membrane was then incubated with rabbit anti-YAP1 monoclonal antibody (1:100, Abcam, Cambridge, MA, USA), or mouse anti-GAPDH monoclonal antibody (1:100, Abcam) as an internal reference for 3 h at room temperature, and then washed by DPBS for 10 min. After that, the PVDF membrane was incubated with mouse anti-rabbit secondary antibody (1:20000, Abcam) for 1 h at room temperature. After washing by DPBS for 15 min, the immune complexes on PVDF membrane was detected using the ECL Western Blotting Kit (Pierce Chemical, Rockford, IL, USA). Image-Pro plus software 6.0 was used to analyze the relative protein expression, represented as the density ratio versus GAPDH.

Dissected retinas were blocked in dPBS (Gibco) supplemented with 1% donkey serum (Sigma‐Aldrich), 0.5% Triton X‐100 (Sigma‐Aldrich) for 48 h at 4°C under gentle shaking. Primary antibodies (Isolectin, 1:200, Thermo Fisher Scientific, Catalog No. I21411; anti‐phospho‐tau clone AT8, 1:500, Thermo Fisher Scientific, Catalog No. MN1020, and conjugated with Alexa‐594 conjugation kit, Abcam, Catalog No. AB269822) were prepared in dPBS with CaCl₂ and MgCl₂, 1% donkey serum, and 0.5% Triton X‐100, and retinas were incubated for 18 h at 4°C. Subsequently, retinas were washed with PBS every 10 min for 2 h and then incubated with DAPI (1:1,000 in PBS, Millipore, Catalog No. 10236276001) for 15 min, and subsequently washed every 15 min with PBS for 45 min in total. Prior microscopy acquisition, retinas were mounted using Vectashield fluoromount (Vector Laboratories, Catalog No. H‐1000‐10).