Cathepsin d

1 to 2μm thick sections from FFPE tissue blocks were cut, dewaxed and pretreated. Immunohistochemical (IHC) stainings were performed with an automated staining device (Dako Autostainer, Dako, Glostrup, Denmark). IHC was carried out with antibodies against ubiquitin (#Z0458, Dako), p62 (#sc-28359, Santa Cruz, CA, USA), LC3B (#0231–100, Nano Tools, Hamburg, Germany), cathepsin B (#sc-6490-R, Santa Cruz), cathepsin D (#sc-6486, Santa Cruz), lamin AC (#2032, Cell Signaling, Danvers, MA, USA) and BRAF^V600E (#ab228461, Abcam, Cambridge, UK). Detailed information on used antibodies and staining protocols are given in S1 Table. Negative controls were included in every run. For negative controls, slides were incubated with non-immune immunoglobulin instead of the primary antibody, carried out in the same concentration as the primary antibody. For positive controls, tumor cases that presented with a specific staining during antibody establishment were included in every subsequent run.
One investigator (DW) evaluated the HE stained slides and the immunohistochemical stains. NI were counted in all three tissue cores per case and total numbers were normalized to one square millimeter.

The expression of PIG7, Pro-Cathepsin B, D, L, Cathepsin B, D, L, Caspase 3, 9, Cleaved Caspase 3, 9, LC3 I/II, ATG5, Beclin-1, Spi2A and Cystatin C were confirmed by Western blot both before and after leukemia cells were transfected with pLenti6.3-PIG7, or pLenti6.3 control vector (either alone or in combination with chemotherapy treatment). The immunoreactive proteins were visualized using the SuperSignal chemiluminescent detection system (Pierce, Rockford, IL, USA) in a similar exposure time. Antibodies against Caspase-3, Caspase-9, LC3B, ATG5, Beclin-1, Cystatin C and MLKL were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Cathepsin B, Cathepsin D, Cathepsin L and β-actin were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibody against PIG7 was purchased from BD Biosciences (San Diego, CA, USA). Antibody against Spi2A was purchased from Abcam (Cambridge, UK).

Primary antibodies for western blotting were VPS13C (HPA043507, 1:500; Sigma-Aldrich, 28676-1-AP, 1:800; Proteintech), phosphoRab10-T73 (230261, 1:500; Abcam), Rab10 (8127, 1:1,000; Cell Signaling), βIII-Tubulin (4466S, 1:4,000; Biolegend), TH (657012, 1:2,000; Millipore), GAPDH (2,118, 1:2,000; Millipore), α-Tubulin (5168, 1:20,000; Sigma-Aldrich), LAMP2 (H4B4, 1:1,000; DSHB), GCase (G4171, 1:1,000; Sigma-Aldrich), Rab7 (137029, 1:1,000; Abcam), HA (3724, 1:2,000; Cell Signaling), Calnexin (1:1,000; Cell Signaling), TOM20 (612278, 1:1,000; BD Biosciences), PEX5 (83020S, 1:500, Cell Signaling), GFP (1544, 1:2,000; Sigma-Aldrich), Myc (2278, 1:2,000; Cell Signaling), cathepsin B (AF953, 1:2,000; R&D systems), cathepsin D (6487, 1:1,000; Santa Cruz), LRRK2 (ab133474, 1:500; Abcam), LRRK2-S935 (ab133450, 1:500; Abcam), EHBP1 (17637-1-AP, 1:1,000; Proteintech), and PPM1H (PA5-26102, 1:1,000; Invitrogen).
Primary antibodies for immunofluorescence were βIII-Tubulin (4466S, 1:300; Biolegends), TH (657012, 1:300; Millipore), and LAMP1 (sc-20011, 1:300; Santa Cruz).
Primary antibodies for PLA were VPS13C (28676-1-AP, 1:100; Proteintech) and Rab10 (ab104859, 1:100; Abcam).

Cells were seed in 6-well plates and treated with different doses of SH003 for 24 hours. Cells were lyzed with RIPA buffer and equal amount of protein (15 μg) in total cell extracts was separated by SDS-PAGE. After transferring to PVDF membrane, the membrane was blocked and blotted with the relevant primary antibodies. Anti-Bax, -Bcl2, -LC3A/B, Cathepsin B, Cathepsin D and -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-cleaved caspase-3, -PARP, -p-mTOR, -p-p70S6K and -p70S6K antibodies purchased from Cell Signaling (Danvers, MA, USA). Anti-LC3B and -p62 antibodies were purchased from Abcam (Cambridge, UK). Analysis of intracellular p62 expression was by flow cytometry using the Alexa Fluor 488-conjugated p62 antibody (BD Biosciences, San Jose CA, USA). Cells were seed in 6-well plates and then treated with 500 μg/ml of SH003 and autophagy inhibitors, such as BaF1 and CQ for 24 hours. After permeabilized with 0.5% Tween-20 in 95% ethanol for 10 minutes, stained with Alexa Fluor 488-conjugated p62 antibody (1:50) for 30 minutes in dark. The data was analyzed by FACSCalibur flow cytometry measuring the green signal by the FL1 channel.

Protein extracts (30μg each) from ALS1 and WT samples were separated by SDS-PAGE and then subjected to Western blotting and immunodetection with the primary antibodies: anti-SOD1, -Cathepsin S, -Cathepsin D, -Cathepsin B (Santa Cruz Biotechnology, CA, USA) [33 (link),41 (link)], -LC3B, (Cell signaling Technology, Danvers, MA, USA), Lamp1 (Sigma Aldrich, St. Louis, MI, USA) and -Actin (Sigma Aldrich, St. Louis, MI, USA), and, with one of the following secondary antibody: Anti-rabbit IgG, HRP-linked Antibody (Cell signaling Technology, Danvers, MA, USA), Anti-mouse IgG, HRP-linked Antibody (Cell signaling Technology, Danvers, MA, USA) Rabbit Anti-Goat IgG Antibody, HRP conjugate (Sigma Aldrich, St. Louis, MI, USA). The ECL^TM Detection System (GE Healthcare, Fairfield, CT, USA) was used for the immunostaining procedures. The same blot was re-probed with different antibodies for comparative analyses.
Densitometry analyses were conducted by the FIJI software (FIJI Life-Line version, v.2015, National Institutes of Health, Bethesda, MD, USA). Relative band intensities were normalized to Actin as internal references. Results are expressed as mean ± SD of three independent experiments.