Fluorescein phalloidin

Cell cultures were fixed [0.1 mol/L, piperazine‐1,4‐bis(2‐ethanesulphonic acid) (Sigma‐Aldrich), pH 6.95, 1 mmol/L ethylene glycol‐bis (2‐oiminoether)‐ N,N,N′,N′‐tetraacetic acid] (Sigma‐Aldrich), 3 mmol/L magnesium sulphate (Sigma‐Aldrich) and 2% paraformaldehyde] at room temperature for 35 min and then permeabilized with 0.1% (v/v) Triton X‐ 100 in PBS. Fixed cultures were then incubated with primary antibodies and secondary antibodies, according to the standard immunochemical approach (Chen et al. 2007). Labelled cultures were then mounted using ProLong Gold Antifade Reagent with 4, 6‐diamidino‐2‐phenylindole (DAPI) stain (Invitrogen), followed by fluorescence microscopy. Anti‐Lgr5 (5 ng/mL, Upstate) and anti‐C. parvum (Zhou et al. 2012) were used. For localization of actin, fluorescein‐phalloidin (Sigma‐Aldrich) was incubated for 30 min at room temperature after incubation with the primary antibodies.

The GFP-HDAC6 expression plasmid was constructed by cloning human HDAC6 cDNA into the pEGFPN1 vector, and the catalytically inactive mutant was generated by site-directed mutagenesis as described (Zhou et al., 2009 (link); Li et al., 2014 (link)). Triethylammonium bicarbonate, formaldehyde-H₂ (28% in H₂O), sodium cyanoborohydride, ammonium hydroxide, trifluoroacetic acid, 4’,6-diamidino-2-phenylindole (DAPI), fluorescein-phalloidin, trichostatin A (TSA), and sodium butyrate (NaB) were purchased from Sigma-Aldrich. Formaldehyde-D₂ (28% in D₂O, Cambridge Isotope Laboratory), formic acid and acetonitrile (Merck), and C₁₈ desalting microcolumns (Nest Group) were from the indicated sources. Tubacin was obtained from Stuart Schreiber (Harvard Medical School). Antibodies against α-tubulin, acetylated α-tubulin, and β-actin (Sigma-Aldrich), HDAC6, HDAC7, HDAC9, Hsp90, Prx I, MYH9, Hsc70, and DNAJA1 (Abcam), HDAC3, HDAC4, HDAC5, HDAC11, and SIRT2 (Santa Cruz Biotechnology), acetylated lysine (Cell Signaling Technology), and GFP (Roche), horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences), and rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were obtained from the indicated sources.

Cells were seeded on 12-well slides (Solarbio, China) and treated as described above. The slides were washed with PBS, fixed in a 4% PFA solution, and rinsed three times with PBS. The cells were permeabilized for 5 min in a 0.5% Triton X-100 solution. After washing with PBS, the cells were labeled with fluorescein phalloidin (1:200, Sigma–Aldrich) for 20 min at room temperature. After washing with PBS, the cells were stained with DAPI at room temperature for 10 min. The cells were viewed using a fluorescence microscope (Olympus, Japan), and the mean optical density was quantified using Image-pro plus 5.1 software.