Axio imager z1 fluorescent microscope

To examine the protein expression and location by immunofluorescent staining, NSCLC cells were seeded onto coverslips in a 24-well plate and left overnight. Cells were then fixed using 4% formaldehyde for 30 min at 25 °C and treated with 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 min. The coverslips were incubated with rabbit anti-ALKBH5, YTHDF1, YTHDF2, YTHDF3, YAP, CTGF, Cyr61, Ki67, Annexin V, Ki67, Edu, Vimentin and mouse anti-E-cadherin monoclonal antibody (Abcam) at 1:200 dilution in 3% BSA. Alexa-Fluor 488 (green, 1:500, A-11029; Invitrogen, USA) and 594 (red, 1:500, A-11032; Invitrogen, USA) tagged anti-rabbit or -mouse monoclonal secondary antibody at 1:1000 dilution in 3% BSA. Hoechst (3 μg/mL, cat. no. E607328; Sangon Biotech Co., Ltd.) was added for nuclear counterstaining. Six pictures were obtained with a Zeiss Axio Imager Z1 Fluorescent Microscope (Zeiss, Oberkochen, Germany). The results were presented as the Mean ± SD.

Six NMRI tumor-bearing mice were treated with vehicle (n = 3) or CA4 (n = 3). Two hours after treatment, the functional perfusion marker Hoechst 33342 (15 mg/kg; iv injection; Sigma-Aldrich) was injected. Mice were sacrificed 2 min later. Five micrometer tumor cryosections were immunostained with a rat monoclonal antibody against CD31 (BD, clone MEC 13.3) and revealed with Alexa568 conjugated anti-rat secondary antibodies (Invitrogen). Tumor sections were imaged using a Zeiss Axioimager Z1 fluorescent microscope equipped with an Apotome module (Zeiss, Wetzlar, Germany).
Estimation of CA4-induced cell death was also obtained from TLT tumor-bearing mice treated with CA4 (n = 7) or vehicle (n = 8). Tumors were excised at day 1 after treatment initiation. Tumors were embedded in Tissue-Tek OCT compound and frozen in liquid nitrogen-cooled isopentane for cryosectioning. Samples were cut into 5 μm sections. The frozen slices were stained with Haematoxylin & Eosin or were probed for cellular death by TUNEL assay using an in situ cell death detection kit (Roche Diagnostics, Belgium). For TUNEL assay, nuclei were also counterstained with Hoechst 33342. Slides were then scanned with a Zeiss Mirax fluorescence microscope. Cell death was quantified using Frida software and expressed as a percentage of the whole tumor area.

The P3 iPSC-RPE cells were grown on coverslips. The cells were rinsed with 1× PBS, fixed in 4% paraformaldehyde for 10 min at 4 °C and then permeabilized in PBS supplemented with 1% of Triton X for 5 min at RT. The cells were cleaned in 1× PBS and subsequently blocked in PBS supplemented with 2% of bovine serum albumin for 30 min at RT. For the immunostaining, the cells were incubated with the primary antibody diluted in a 2% bovine serum albumin in 1× PBS for 2 h at RT. The cells were washed 4 times for 5 min in 1× PBS and incubated between 45 and 60 min with the corresponding secondary antibodies. The cells were washed 3 times for 5 min in 1× PBS. Finally, the slides were mounted in Vectashield without DAPI (Life Technologies, Carlsbad, CA, USA). The cells were imaged on a Zeiss Axio Imager Z1 Fluorescent microscope (Zeiss, Aalen, Germany) and analyzed using Fiji software. The employed antibodies and their combinations were indicated in Table S2.

NSCLC tumor cells were seeded on glass coverslips, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with 0.5% Triton X-100 (Biosharp, China) for 10 min at room temperature. The slides were then processed using a RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, China). The corresponding FISH Probe Mix was also designed by RiboBio Co. The experiment was repeated three times in A549 cells. Images were obtained with a Zeiss Axio Imager Z1 Fluorescent Microscope (Zeiss, Oberkochen, Germany).