α tubulin

Protein extract preparation and western blot analysis were performed as previously described^{47 (link)}. Primary antibodies used were: PRDX2 (ab10367; Abcam), SOX11 (MRQ-58; Cell Marque), p-AKTS473 (Cell Signaling), AKT (sc-1618; Santa Cruz Biotechnology), p-MAPK T202/Y204 (p-ERK 42/44; Cell Signaling), ERK1/2 (sc-94; Santa Cruz Biotechnology) and α-tubulin (Abcam).

The ADSCs and MDA-MB-231 cells collected from the co-culture system or GDF15-treated MDA-MB-231 cells were lysed with RIPA lysis buffer. Protein concentrations determined using Bio-Rad protein assay (BIO-RAD, Hercules, CA, USA). Equal amounts of total proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane (IBFP0785C, Merck Millipore, Burlington, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated with primary antibodies at 4 °C overnight. Primary antibodies used in western blotting included β-actin (GTX109639, GeneTex, Hsinchu, Taiwan), α-tubulin (GTX112141), ZEB1 (GTX105278), Snail (GTX82509), ZO-1 (GTX108627), Nanog (GTX100863), Oct4 (GTX101497), β-catenin (ab16051, Abcam, Cambridge, MA, USA), GDF15 (PAB31426, Abnova, Taipei, Taiwan), phosphor-AKT (4060, Cell Signaling, Danvers, MA, USA), and AKT (4691, Cell Signaling). Immunoreactive proteins were detected after incubation with horseradish peroxidase-conjugated secondary antibody (31430, Thermo Scientific) for 1 h at room temperature. The immunoblots were visualized using chemiluminescence reagent (WBKLS0500, Merck Millipore) and quantified by ChemiDoc XRS+ imaging system (BIO-RAD).

Immunofluorescence was performed as previously described59 (link). Antibodies were purchased from Sigma (α-tubulin, γ-tubulin, centrin and Flag), Abcam (γH2AX) and Invitrogen (Alexa Fluor 568-conjugated IgG secondary antibody). Of note, cells were incubated at 4 °C for 1 h before permeabilization and fixation for centriole staining9 (link).

In vitro, UTD1, Paclitaxel, and 5-Fluorouracil (5-FU) (stock solution 10 mg/ml) were diluted in complete medium according to required concentration before use. Complete medium was used as control. In vivo, drugs were diluted in 0.9% sodium chloride aqueous solution before administration. And 0.9% sodium chloride aqueous solution was used as control. RPMI-1640 medium, FBS, and crystal violet were purchased from SIGMA. PI/RNase Staining Buffer and FITC Annexin V Apoptosis Detection Kit were purchased from BD Biosciences. Paclitaxel, 5-FU, Z-VAD-FMK, SP600125, and Trolox were purchased from Med Chem Express (Shanghai, China). Antibodies against cyclinB1, CDC2, P21, PARP, Cleaved caspase-3, cytochrome C, phospho-JNK, Ki-67, Drp1, Mitofusin-2, and secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). CyclinA2, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-Tubulin, and secondary antibody were purchased from Abcam (UK).

HIV-infected cells were stained with the following primary antibodies: human anti-HIV-1 Gag p24 (37G12: Centre for AIDS Reagents, NIBSC, UK, supported by EURIPRED (EC FP7 INFRASTRUCTURES-2012 -INFRA-2012-1.1.5.: Grant Number 31266; Polymun) conjugated to Aberrior STAR 635P dye (Abberior GbmH) by NHS chemistry. Conjugation was performed by incubation of concentrated 37G12 (2–4 mg/ml), 0.2 M NaHCO₃ (pH 8.3) and Aberrior STAR 635P NHS Ester (3:1 molar dye:antibody ratio) in PBS for 1 h at RT, followed by removal of unbound dye by filtration through Spin-X UF 500 50 k MWCO columns (Corning). Antibody concentration and degree of labelling was determined by NanoDrop. CD8⁺ T cells were identified using a mouse anti-human CD8α (clone 37,006, R&D Systems) or rabbit anti-human CD8α (Abcam). Immunological synapses were imaged using antibodies against perforin (clone dG9; BioLegend), α-tubulin (Abcam) and Zap70 (Abcam) or the Alexa Fluor 488 phalloidin stain (Molecular Probes). All unconjugated mouse and rabbit primary antibodies were labelled with a goat secondary antibody coupled to Alexa Fluor 488 or 568 (Life Technologies).

The tissue was harvested at indicated time points and lysed in a lysis buffer (0.5% Nonidet P-40, 10 mM Tris, pH 7.4, 150 mM Nacl, 1 mM EDTA, 1 mM Na₃VO₄) with a protease inhibitor (1 mM PMSF). BCA assay was used to quantify protein level. Cell lysates (30 μg protein) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Immobilon; Millipore, Bedford, MA). For immunoblotting, primary antibodies against α-SMA, TGF-β, α-tubulin and β-actin (1:1000) (Abcam, Cambridge, UK) and IgG-HRP secondary antibody (1:2000) were used. Chemiluminsescent signals were acquired using Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Densitometric analysis was performed using Image J 5.0 software. Four biological replicates were performed.