Alexa fluor 488

As described by our previous studies (13 (link), 14 (link), 16 (link)), to evaluate the effect of rapamycin on damage to oral cancer cells, a H2A.X flow cytometry was performed. In addition, after treating Ca9-22 cells with the studied concentrations of rapamycin, they were trypsinized and then fixed with 75% ethanol for 15 min. The centrifugation of the samples was carried out to eliminate the fixative solution. Afterward, a permeabilization solution containing 1% BSA/0.2% Triton/1X PBS was added to the cells and they were then incubated in the dark at 4°C overnight with the first phospho-histone H2A.X (Ser139) monoclonal antibody from Santa Cruz Biotechnology at a dilution 1/100, washed twice with PBS and incubated with the secondary antibody conjugated to Alexa Fluor 488 from Santa Cruz Biotechnology in a 1:100 ratio for 1h before analyzing them with the BD flow cytometry system (BD FACS Canto II) and the percentage of positive cells was calculated. This experiment was repeated three times.

Nuclear translocation of Nrf2 was examined by immunofluorescence staining. HUVECs were fixed with 3.7% paraformaldehyde in PBS for 15 min followed by treating permeabilization buffer (0.1% Triton X-100 in PBS) for 10 min. HUVECs were incubated with blocking solution (2% bovine serum albumin) for 30 min at room temperature. After washing with PBS, HUVECs were treated with anti-Nrf2 antibody (1:200 dilution, Santa Cruz Biotechnology) at 4 °C for overnight. The secondary antibody labeled with Alexa Fluor^® 488 (1:500 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) was treated for 1 h. Nuclei were counterstained with 2 μg/mL Hoechst 33342 for 10 min. The stained HUVECs were visualized using a fluorescence microscope.

The following antibodies were used for immunofluorescence: mouse monoclonal anti-human/mouse proinsulin biotinylated antibody (1:200; R&D Systems (Minneapolis, MN, USA), #BAM13361), mouse monoclonal anti-calnexin antibody (1:100; Novus Biologicals (Englewood, CO, USA), #NB300-518), rabbit polyclonal anti-GM130 antibody (1:200; Novus Biologicals, #NBP2-53420), guinea pig polyclonal anti-insulin antibody (1:50; GeneTex (Zeeland, MI, USA), #GTX27842). Secondary antibodies were from ThermoFisher Scientific: anti-streptavidin AlexaFluor-568 (1:200; #S11226), AlexaFluor-488 (1:200; #A11001 and #A11073) and AlexaFluor-568 (1:200; #A10042).
The antibodies used for immunoblotting were the following: mouse monoclonal anti-actin antibody (C4) (1:500), rat monoclonal anti-GRP78/Bip (1:250) and mouse monoclonal anti-vinculin (1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA), rabbit polyclonal anti-Phospho-eIF2α (1:500), rabbit polyclonal anti-eIF2α (1:500) and mouse monoclonal anti-Chop (clone L63F7) (1:200) from Cell Signaling (Danvers, MA, USA), rabbit polyclonal anti-Atf4 (1:1000) from GeneTex. Secondary antibodies conjugated with HRP were from Biolegend (anti-mouse and anti-rabbit) and from Santa Cruz Biotechnology (anti-rat). Standard blocking conditions (5% milk in TBS-T) were used throughout, except when anti-P-eIF2α antibody was used, and 1% BSA in TBS-T was utilized.

Normal human eyeballs (n = 3), from deceased individuals, were obtained from the archive of Ocular Pathology Service, the Chinese University of Hong Kong. The localization of RRAGA protein was determined by immunofluorescence analysis. Whole eyeballs were fixed in 10% formalin and embedded in paraffin. Paraffin sections (5 μm thick) were first de-waxed and re-hydrated in sequential incubation in xylene and ethanol. Antigen was retrieved in Tris-EDTA buffer at 80°C for 20 minutes. After permeation and blocking in 0.425% saponin, 0.0015% Triton-X 100, 0.0015% Tween-20, 1% bovine serum albumin and 1% normal goat serum, eyeball sections were labeled with primary rabbit polyclonal antibodies against RRAGA at 4°C for 16–18 hr and secondary antibodies against rabbit IgG, conjugated with Alexa Fluor 488 (Santa Cruz Biotechnology), at room temperature for 1 hr. Fluorescence was visualized under a fluorescence microscope (Eclipse Ni-U, Nikon).

Di-rhaminolipid with chemical structure in Fig. S1 was a gift from Zijing Bio. Inc. (Huzhou, China) with purity >99%. TGF-β1 was purchased from Peprotech (Rocky Hill, NJ). Collagenase I, Dispase, Calcein-AM, propidium iodide (PI), Alexa Fluor 488 phalloidin, Fura-2-AM, DMEM medium were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Primary antibodies (anti-α-SMA and anti-β-Tubulin) and secondary antibodies (goat-anti-mouse, HRP and Alexa Fluor 488) were purchased from Santa Cruz Biotech. Inc. (Dallas, TX). Fetal bovine serum (FBS) was obtained from Gibco (Invitrogen Co. Ltd, Canada). Collagen (type I, from rat tail) was purchased from Biot Biology Inc (Wuxi, China). Sircol insoluble collagen assay kit was purchased from Biocolor Ltd. (Northern Ireland, UK). LDH assay kit was purchased from Saike Biotech. Inc (Ningbo, China). The remaining chemicals were obtained from local chemical suppliers and were all of reagent grade.

For BrdU incorporation assay, cells were labeled with 10 μm BrdU (Roche, Indianapolis, IN, USA) for 2 h, fixed with 4% para-formaldehyde, and immunostained with anti-BrdU antibody (Roche) followed by staining with Cy™3-conjugated goat anti-mouse IgG (115-165-146; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and counter-stained with DAPI. BrdU-positive cells were scored under a fluorescent microscope and presented as the percentage of BrdU-positive nuclei over total number of nuclei counted. At least 300 nuclei were counted. For immunofluorescence, cells were fixed with 4% paraformaldehyde, immunostained with primary and secondary antibodies in 4% BSA, and counter-stained with DAPI. Antibodies used include anti-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse Alexa Fluor 488 (A11001; Santa Cruz Biotechnology). Cell images were recorded with an Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany) and analyzed with axiovision 3.1 software (Carl Zeiss).