Anti cathepsin b

The following chemicals were purchased from Sigma Aldrich: silver nitrate (AgNO₃), sodium borohydride (NaBH₄), hydrogen tetrachloroaurat(III) (HAuCl₄), L‐ascorbic acid, cetyltrimethy‐ lammonium bromide (CTAB), and Fluorescein isothiocyanate (FITC); Fetal calf serum (FCS) was purchased from Gibco; CCK‐8 Kit was purchased from Dojindo Laboratories; Annexin V‐FITC/PI Apoptosis Detection Kit was purchased from Becton, Dickinson and Company; LysoTracker‐Red was purchased from Invitrogen; TNF‐α ELISA kit was purchased from R&D SYSTEMS; fluorochrome‐conjugated secondary antibody (Invitrogen); JC‐1 Mitochondrial Membrane Potential Assay Kit was purchased from Cayman Chemicals; The following antibodies were purchased from Cell Signaling Technology: anticathepsin B, anticathepsin D, anticaspase 8, anticaspase 9, anticaspase 3, anti‐RIP1, anticytochrome c, anti‐LAMP‐2; The following inhibitors were purchased from Selleck Chemicals: Necrosis inhibitor necrostatin‐1, caspase inhibitor Z‐VAD‐FMK, cathepsin B inhibitor CA‐074‐Me; caspase 8 inhibitor Z‐IETD‐FMK; 18.2 MΩ cm Ultrapure water produced by a Millipore Milli‐Q Plus Water Purification System from Millipore was used in all experiments. All chemicals were used as received without further purification.

Immunohistochemistry and H&E staining were performed on cryo- or paraffin-embedded tissue sections. Primary antibodies were anti-STEM121 (1:100; Takara Bio Inc., Y40410), anti-cleaved Caspase-3 (1:200; Cell Signaling Technology, 9661), anti-cathepsin B (1:200; Cell Signaling Technology, 31718S), anti-γH2Ax (1:500; Cell Signaling Technology, 2577S), anti–phospho-Histone H3 (1:200; Cell Signaling Technology, 9706S), and anti-CD44 (1:200; Abcam, ab243894). Secondary antibodies conjugated to Alexa Fluor dyes (488, 555, and 647) at a dilution of 1:200 were used. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling kit (Sigma-Aldrich, cat. no. S7110,) was used for apoptosis quantification. DAPI (Sigma-Aldrich, cat. no. D9564) was used for nuclear counterstain. Images were acquired using a Leica SP8 Lightning Confocal DMI6000 microscope. Images were analyzed using Imaris software.

Total protein extracted from the medulloblastoma cells was separated by SDS-PAGE electrophoresis, blotted onto a PVDF membrane (Merck Millipore, Berlington, MA, USA) and probed with the primary antibody as per the manufacturer’s protocol. The images were captured using the ChemiDoc gel imaging system (Biorad Hercules, CA, USA) or by autoradiography. The captured images were quantified using the Image Lab software (Bio-Rad, Hercules, CA, USA) or ImageJ software (imajeJ.nih.gov.in). The antibodies used for the Western blotting experiments are listed below.I.

Anti-LC3B (#2775), anti-p62/SQSTM1 (#8025), anti-Cathepsin D (#2284), anti-Cathepsin B (#31718) and, anti-IGF2R (#14364) antibodies from the Cell signaling technology, Boston, MA, USA.

II.

Anti-GAPDH antibody (SC 47724) from Santa Cruz Biotechnology, Dallas, TX, USA.

III.

Anti-Histone H3 (acetyl K9) antibody (ab10812) from Abcam, Cambridge, UK.