Anti atf3

Western blot assay was performed as the previous study described [48 (link)]. Total proteins were extracted using RIPA buffer (#FD011, Hangzhou Fude Biological Technology) and quantified by BCA protein assay kit (#P0011, Beyotime). In total, 1 μg of proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (#IPVH00010, Merck Millipore). After blocked with 5% Non-Fat Milk and incubated with specific antibody at 4 °C overnight, followed by HRP-conjugated secondary antibody incubation, the membrane was imaged with imager (Biorad ChemiDoc MP, Biorad). The antibodies used in this study were listed: anti-GAPDH (#5714, Cell Signaling Technology), anti-METTL1 (#ab157097, Abcam), anti-E-cadherin (#20874-1-AP, Proteintech), anti-N-cadherin (#66219-1-AP, Proteintech), anti-VIMENTIN (#10366-1-AP, Proteintech), anti-MMP2 (#10373-2-AP, Proteintech), anti-CDK4 (#11026-1-AP, Proteintech), anti-P16 (#18769, Cell Signaling Technology), anti-ATF3 (#ab254268, Abcam).

U87 and primary IC glioma cells were cultured in DMEM containing 0.4% FBS. Supernatant of JS-K treated cells was collected and frozen at −20 °C. Cells for whole cell lysates were cultured in 10% FBS. Equal amounts of protein (4 μg of supernatant, 20 μg of lysate) were applied on 10% SDS-polyacrylamide gels and electrophoresed (BioRad, Munich, Germany). Proteins were blotted on PVDF-membranes by wet blotting (BioRad, Munich, Germany). Epitopes were blocked with 5% non-fat milk in tris-buffered saline with 0.05% Tween20 for 1 h at room temperature (RT). Blots were incubated with primary antibodies anti-ATF3 (ab87213 1 : 1000 Abcam, Cambridge, UK), anti-MMP2 (#4022 1 : 1000 Cell Signaling Technology, Inc., Danvers, MA, USA), anti-MMP7 (#MAB9071 1 : 1000 R&D System, Inc., Minneapolis, USA), anti-MMP9 (#3852 1 : 1000 Cell Signaling Technology, Inc.) anti-TIMP3 (#D74B10 1 : 1000 Cell Signaling Technology, Inc.), anti-GAPDH (1 : 10000 Abcam) overnight at 4 °C. After incubation with secondary antibodies goat anti-rabbit/mouse (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at RT proteins were visualized by enhanced chemiluminescence (BioRad). For loading control, a Coomassie-stained SDS-polyacrylamide gel was used for whole protein in the supernatant and GAPDH was used for the whole cell lysate.

Mice were deeply anesthetized with isoflurane and perfused through the ascending aorta with PBS, followed by 4% paraformaldehyde in 0.16 mol/L phosphate buffer. DRG sections were cut at 14 μm on a cryostat. ISH was applied with an RNAscope™ Fluorescent Multiplex Assay kit and probe targeting mouse Tmem63a from Advanced Cell Diagnostics strictly following the suggested procedure. After finishing ISH, the sections were incubated overnight at 4°C with the following primary antibodies: anti-SP (guinea pig, 1:1000; Neuromics), anti-CGRP (rabbit, 1:1000, Sigma), anti-TH (rabbit, 1:1000, Millipore), and anti-NF200 (mouse, 1:1000, Sigma), followed by Cy3- or FITC-conjugated secondary antibodies (1:200; Jackson Immuno Research Laboratories Inc.) or FITC-conjugated IB4 (10 μg/mL, Sigma-Aldrich). Sections for immunostaining were incubated with anti-ATF3 (rabbit, 1:1000, Abcam), anti-CD68 (rat, 1:200, Biolegend), and anti-β-tubulin III (mouse, 1:1000; Chemicon) followed by Cy3- or FITC-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories Inc.), or DAPI (1:1000; Invitrogen), or Nissl (1:1000; Invitrogen). Sections were mounted and examined under a Nikon fluorescence microscope or Olympus FV1000 confocal laser scanning microscopy.