C cas 3

Rats (n = 5/group) were sacrificed without pain. Brain tissue was fixed in formaldehyde, embedded in paraffin, and sectioned with a thickness of 5 mm. Nonspecific antigen binding was blocked with normal sheep serum for 60 min. The sections were separately incubated with primary rabbit antibodies against LC3 (1:200; Cell Signalling Technology, Danvers, MA, USA) and CCAS3 (1:200; Cell Signalling Technology, Danvers, MA, USA) at 4°C overnight. After washing with PBS, the sections were incubated with Rhodamine (TRITC)-conjugated goat antirabbit IgG (H+L) secondary antibody (1:500; proteintech) at room temperature for 1 h in the darks and then washed three times with PBS. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were observed at 200× magnification under a fluorescence microscope (OLYMPUS 905). Outcomes are shown as optical density (OD). In our present study, immunofluorescence analysis demonstrated the expression difference of LC3 in the ipsilateral hippocampus of the rats in different groups.

IHC was performed on FFPE cores of SCLC tumours or CDX tumours, and staining was done on 4 μm sections using recombinant anti-GUCY1B1 antibody (Abcam, ab154841) at 6.76 µg/ml, pHH3 (Millipore, 06-570) at 0.4 µg/ml, and cCas3 (Cell Signaling Technology, 9661) at 0.2595 µg/ml diluted in Bond Primary Antibody Diluent (Leica Biosystems, AR9352). Heat-induced epitope retrieval (HIER) was performed using BOND Epitope Retrieval Solution 1 (Leica Biosystems, AR9961) for 20 min and staining was carried out using the Leica Bond Max Platform performing standard protocol F with Bond Polymer Refine Detection (Leica Biosystems, DS9800). Scanning of the stained slides was performed using a Leica SCN400. Digitally scanned slides were analysed using HALO software v2.3 (Indica Labs).Tumour regions were defined within each sample using a machine learning classifier. Tumour cells were detected and classified as positive or negative based on IHC thresholds using the Area Quantification algorithm. Expression level was quantified as percentage positive tumour within the sample.

A total of 30 µg of protein was separated using 10% SDS-PAGE and transferred to a PVDF membrane, which was then blocked with 5% w/v BSA. Membranes were probed with the indicated primary antibodies including rabbit polyclonal antibodies against Cytc, c-cas3, Bcl-2, phosphorylated (p)-AMPKα (Thr172), p-PI3K, PI3K, p-AKT, AKT, p-p53, and p53 (Cell Signaling Technology, USA) along with mouse monoclonal antibodies against Bax and AMPK (Proteintech, China), and GAPDH was used to normalize protein expression. Appropriate secondary antibodies conjugated to horseradish-peroxidase (HRP) were then added and incubated for 1 h. The antigen-antibody complex was detected using an enhanced chemiluminescence reagent (Millipore, USA). The gray intensity of the bands on the western blots was analyzed using the ImageJ software (NIH, Bethesda, USA).

The following antibodies were used in the current study: α-SMA (SC-53142; Santa Cruz Biotechnology, Dallas, TX, USA); Fibronectin (SC-69681; Santa Cruz Biotechnology); Col1α1 (SC-293182; Santa Cruz Biotechnology); KLF10 (MA5-38047; Invitrogen, Carlsbad, CA, USA); ATF3 (#18665; Cell Signaling Technology, Dallas, TX, USA); Phospho-Smad2 (Ser465/467) (#3108; Cell Signaling Technology); Phospho-Smad3 (Ser423/425) (#9520; Cell Signaling Technology); SMAD2/3 (610842; BD Biosciences, San Jose, CA, USA); FLAG (F1804; Sigma-Aldrich, St. Louis, MO, USA); GAPDH (MAB374; Millipore, St. Louis, MO, USA); β-actin (SC-47778; Santa Cruz Biotechnology); PARP (#9542, Cell Signaling Technology); and C-Cas3 (#9664, Cell Signaling Technology).

CBD was obtained from Sigma. CBD dissolved in absolute Ethanol (EtOH) was stored at −20 °C. CHX was purchased from Merck Milipore (Darmstadt, Germany). MG132 was purchased from Sigma. Antibodies against c-PARP, c-Cas3, -Cas8, and Cas9, Bid, Bax, p53 upregulated modulator of apoptosis, XIAP, Bip, GRP94, PERK, p-PERK, phospho-IRE1α, p-IRE1α, ATF6, CHOP, ubiquitin (Ub), and cytochrome c oxidase subunit I were purchased from Cell Signaling Technology (Danvers, MA, USA). Protein G PLUS-Agarose beads and antibodies against B-cell lymphoma-extra-large, NDUFA9, succinate dehydrogenase complex flavoprotein subunit A, RieskeFeS, and adenosine triphosphate synthase subunit α were purchased from Santa Cruz Biotechnology. β-Actin was purchased from Sigma. Antibody against p8 was purchased from abcam (Cambridge, UK). The secondary antibodies anti-mouse-IgG-horseradish peroxidase (HRP) and anti-rabbit-IgG-HRP were purchased from Cell Signaling Technology.

The peri-infarct region of the stroke-damaged brain was used for immunofluorescence analysis. Briefly, the brain sections were incubated with blocking buffer (1× PBS/7.5% house serum/0.3% Triton X-100) for 1 h at room temperature. They were then incubated overnight with the following primary antibodies (diluted in antibody dilution buffer, 1× PBS/1% bovine serum albumin/0.3% Triton X-100) at 4°C: ionized calcium binding adaptor molecule 1 (Iba1) (1:500, 019-19741; Wako Chemicals, Richmond, VA), CD68 (1:500, sc-135872, Santa Cruz Biotechnology), NeuN (1:500, MAB377, Millipore, Billerica, MA, USA), SREBP1 (1:100, NB100-74542, Novus Biologicals), pNFkB1 (1:100; PA5-37658, Invitrogen), and c-Cas3 (1:50; 9661, Cell Signaling Technology). The sections were washed with PBST and incubated with fluorescent secondary antibodies (A11001 or A11037; Invitrogen) for 2 h at room temperature. Sections were mounted using a mounting medium with DAPI (H-1200-10, Vector Laboratories), and images were captured using a fluorescence microscope (Zeiss Imager M1, Carl Zeiss) and fluorescence K1-fluo confocal microscope (#SMTH-0607-100T; NANOSCOPE Systems, Daejeon, Republic of Korea). Positive cells and integral optical density (IOD) were quantified in a blinded manner.