Er tracker red dye

The original strain BY4741 (CK) and the candidate gene knockout strains were cultured overnight in a constant temperature shaker incubator at 30°C and 200 r/min, and the cells labeled as 0 h were collected at this time. Then, in YPD + G418 liquid medium, the original strain BY4741 and the gene knockout strain broth cultures that had been incubated overnight were separately added to YPD + G418 liquid medium containing 1.6 g/L CA (OD₆₀₀ = 0.8), and cultured for another 3 h at 30°C and 200 r/min, and the cells labeled as 3 h were collected. The collected 0 h and 3 h broths were separately added to 1.5 mL EP tubes (OD₆₀₀ = 1.0), and the centrifuged broths were stained with the stains (thawed in advance)-Diaminophenylindole (DAPI) (purchased from Shanghai Shenggong Biotechnology Co., Ltd.), Mito Tracker Green FM, ER-Tracker Red dye, Yeast Vacuole Membrane Marker MDY-64 (all purchased from Thermo Scientific), and 2′7′-DCFdiacetate (purchased from Sigma), respectively. Afterwards, changes insubcellular structures were observed in real time using a fluorescence microscope equipped with DIC, GFP, Rhod, and DAPI filters, and the accumulation of ROS, chromatin disorder, mitochondrial damage, endoplasmic reticulum damage, and vacuole damage in the cells were statistically analyzed (Li et al., 2023 (link); Wang et al., 2023 (link)).

To localize the different charged residue mutants of TMD9 of Hgt1p that were created, indirect immunofluorescence was performed using a published protocol modified as described earlier (Kaur et al. 2009 (link)). For staining of ER (endoplasmic reticulum), live yeast cells were incubated with the ER-Tracker TM Red dye (BODIPY TR glibenclamide; Invitrogen, USA) according to the manufacturer’s instructions and by other published literatures (Feng et al. 2010 (link); Mandal et al. 2012 (link); Rawal et al. 2013 (link)). Images were obtained with an inverted LSM510 META laser scanning confocal microscope (Carl Zeiss) fitted with a Plan-Apochromat ×100 (numerical aperture, 1.4) oil immersion objective. The 488-nm line of an argon ion laser was directed over an HFT UV/488 beam splitter, and fluorescence was detected using an NFT 490 beam splitter in combination with a BP 505 to 530 band pass filter. ER-Tracker fluorescence was detected at Ex/Em 587/615. Images obtained were processed using Adobe Photoshop version 5.5

The transfected cells on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were then permeabilized in 0.1% Triton X-100/PBS for 10 min, followed by 1 h of treatment with a blocking solution consisting of normal goat serum (1:200, Abcam, ab7481) in PBS. The pan-SOD1 antibody (1:400, Genetex; GTX100554) solution, diluted in the blocking solution, was added to the cell solution overnight at 4 °C. After washing with PBS, we incubated the cells with Alexa Fluor 488 antibody (Goat anti-Rabbit IgG, 1:500, Thermo Fisher Scientific, #A-11008) at 4 °C for 6 h. The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI), and the endoplasmic reticulum (ER) was stained using ER-Tracker^TM Red dye (Invitrogen) for 10 min. After washing the coverslips three times with PBS, we mounted the coverslips with mounting solution (H-5501; Vector Laboratories) for fluorescence microscopy (Zeiss).
For inclusion positive cells counting, immunofluorescence images with SOD1 antibodies were counted in randomly selected fields. Depending on SOD1 staining, inclusion positive cells were counted based on the strong intensity of SOD1 and expressed as a percentage of total cells counted.

The c-terminal (aa 175–239) and n-terminal (aa 1–174) fragments of YFP were PCR amplified from pEYFP-Pds1Δdestruction box (383) which was a gift from Jonathon Pines (Addgene plasmid # 39848), sequenced, and cloned as an in-frame fusion to pEGFP-N1 containing NAF-1 or mNT [19 (link)]. The linker sequence DPRSIAT was used for connecting the mNT/NAF-1 proteins to the YFPc/YFPn fragments. HEK-293 cells plated in EMEM (ATCC 30–2003) with 10% FBS on coverslips coated with collagen (A1048301, gibco) were co-transfected with the different fusion protein constructs (S1A Fig), using the GeneJuice Transfection Reagent (70967–3, Novagen; [4 (link)–6 (link)]). Following staining with the ER-Tracker Red Dye (E34250, Invitrogen) or MitoTracker Deep Red FM (M22426, Invitrogen), complete FluoroBrite DMEM (A18967-01, Gibco) medium was used to replace the RPMI medium 1640, and confocal images were obtained using a Zeiss LSM 710 confocal microscope. YFP, Mito-Tracker, ER-Tracker and DAPI were excited by a 488nm Argon, 561nm diode, 633nm diode and 405nm Diode laser respectively, and their fluorescence was detected at 508-604nm, 635-722nm, 585-700nm and 400-585nm, respectively.

For live cell membrane protein labelling, Piezo1^−/− HEK293T cells were plated on 96 well clear bottom plate (ThermoFisher) coated with 0.1 mg/ml of Poly-L-Lysine (Sigma). Cells were transfected with pIRES-GFP constructs carrying WT or mutant Piezo1 (125 ng cDNA per well) with PEI. Sixty to 72 h after transfection, live labelling was performed by incubating the cells with anti-HA (Sigma, 1:100) antibody for 20 min at 37°C. The cells were then washed with DMEM six times before being incubated with Alexa Fluor 555 anti-mouse secondary antibody (1:200) for 15 min at room temperature (22°C). Cells were washed again 5 times with DMEM and twice with phosphate buffered saline (PBS), then fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. PFA was washed off and replaced with PBS before confocal analysis.
For co-labelling of Piezo1 and the endoplasmic reticulum (ER), HeLa cells were transfected with WT or mutant Piezo1 fused with GFP. After transfection for 60–72 h, cells were washed with PBS twice then incubated with 1 μM of ER-Tracker Red dye (Invitrogen) for 20 min at 37°C. The cells were washed again with PBS three times and fixed with 4% PFA for 20 min at room temperature. PFA was then replaced with PBS, and the ER or Piezo1-GFP signals were visualized using confocal microscopy (Zeiss LSM 700 inverted).

Antibodies against p65 (C-20), SNF2H (H300), Tubulin (TU-02) and HA-probe (HA.C5) were acquired from Santa Cruz Biotechnology. Anti-JMJD8 and PDIA3 antibodies were purchased from Abnova. The anti-calnexin antibody was purchased from Abcam. Antibodies against EEA1 (C45B10), RCAS1 (D2B6N), AIF (D39D2), Kinectin 1 (D5F7J) and Lyric/Metadherin (MTDH) (2F11C3) were purchased from Cell Signaling Technology. LysoTracker® Red DND-99 and ER-Tracker™ Red dye were purchased from Invitrogen.