Nf κb

Cells were treated with doxycycline at 1 g/ml, with 15 nM Actinomycin D (ActD, Invitrogen) for the indicated times in each case. Total protein from MCF7ras, HCC1806 or HS578t were obtained using Pierce RIPA buffer reagent (ThermoFisher), including a cocktail of proteases inhibitors and phosphatases inhibitors (Roche). 20 µg of protein were loaded into SDS-PAGE gels with a gradient from 4 to 15% (Bio-Rad). Proteins were transblotted in a wet-transfer system (Bio-Rad) to PVDF membranes (Immobilon-P, Millipore). Next, membranes were blocked with 5% non-fat milk in TBS and 0,01% Tween-20 (v/v). Membranes were blotted with antibodies against Bim, Bax, Bcl2, Bad, Bid, Puma, PARP, cleaved PARP, caspase 7, cleaved caspase 7, caspase 9, cleaved caspase 9, EGFR, HER2, HER3, AKT, pAKT 473, p42 p44 MAPK, pMEK1/2, LRP6, P-LRP6, Wnt5 a/b, Axin, NF-κB, pNF-κB, IKKα, IKKβ, IκBα, GAPDH (1:1000 from Cell Signaling) and LACTB and Caspase 8 (1:1000, ProteinTech). Signal was detected with enhanced chemiluminescence (ECL) using Azure c600 Western blot Imaging system.

Mice were trans-cardiacally perfused with 4% paraformaldehyde. Their brains and colons were post-fixed with 4% paraformaldehyde for 4 h, cytoprotected in 30% sucrose solution, freezed, cut using a cryostat (Leica, Nussloch, Germany), and immunostained according to the method of Jang et al. (11 (link)). Briefly, the sections were washed with phosphate buffered saline, blocked with normal serum, incubated with antibodies for Iba1 (1:200, Abcam), LPS (1:200, Millipore), NF-κB (1:100, Cell Signaling), CD11b (1:200, Abcam), CD11c (1:200, Abcam), and/or NeuN (1:200, Millipore) overnight, and treated with the secondary antibodies for 2 h. Secondary antibodies conjugated with with Alexa Fluor 488 (1:200, Invitrogen) or Alexa Fluor 594 (1:200, Invitrogen) were then treated to visualize. Nuclei were stained with 4′,6-diamidino-2-phenylindole, dilactate (DAPI, Sigma). Immunostained tissue slices were scanned with a confocal laser microscope.

Phycocyanin (electrophoretic purity) was isolated and purified from Spirulina platensis according to the protocols reported previously with minor modifications56 (link). Isolated phycocyanin was dissolved at a concentration of 1 mM in PBS (pH 7.4) as a stock solution and kept at −80 °C. It was diluted with RPMI-1640 medium (Gibco, 23400-021) before each experiment to keep the final concentration the solvent less than 5% (v/v) throughout the study.
Primary antibodies to MAP-LC3, cathepsin D, LAMP-1, p70S6K, p-p70S6K and Histone H3 were available from Santa Cruz Biotechnology (USA). Primary antibodies against procaspase, caspase, PARP and GAPDH were purchased from Beyotime Institute of Biotechnology (China). Primary antibodies for Beclin 1, Akt, p-Akt, P38, p-P38, Erk1/2, p-Erk1/2, JNK, p-JNK, mTOR, p-mTOR and NF-κB were from Cell Signaling Technology (USA). Horse radish peroxidase-conjugated secondary antibodies were purchased from sigma (USA). Fluorescence-conjugated secondary antibodies were from Invitrogen (USA). Chloroquine (Chlor) were purchased from sigma (USA). NF-κB SN50 and PD98059 were from Merck Millipore (USA). Caspase 3 siRNA, Beclin 1 siRNA and control siRNA was obtained from Cell Signaling Technology, Inc. (CST, USA).

Proteins were isolated from treated THP-1 cells or cutaneous excised tissues using radioimmunoprecipitation buffer with protease inhibitors (Sigma). Protein concentrations were determined by a Bradford assay (Pierce). The total protein (30 μg) of each sample was subjected to SDS-PAGE and immunoblotting with the desired antibodies against TLR4 (Abcam), STAT3 (Cell Signaling), p-STAT3 (Cell Signaling), AKT (Cell Signaling), p-AKT (Cell Signaling), NF-κB (Cell Signaling), p-P65 (Cell Signaling) and β-actin (Abcam), as previously described [27 (link)].