Hoechst 33342 solution

For confocal analysis, DC2.4 cells were seeded for 48 h in eight-well μ-slides (Ibidi) prior to treatment with 2.5 µM fluor-peptide (4 h), then extensively washed with phosphate-buffered saline (PBS) to remove surface-bound peptide and fluorophores. Samples were stained with Hoechst 33342 Solution (BD Biosciences) and CellBright Steady Membrane 550 (Biotim) in complete RPMI Lysotracker Green DND-26 (Invitrogen). Cells were then washed and imaged in Live Cell Imaging Solution (Invitrogen). Images of DC2.4 cell lines were acquired using an Olympus Fluoview FV1200 microscope equipped with a 100× objective, and optimum lasers and filter sets. The images within each dataset were acquired under identical settings and subsequently processed using Fiji image analysis software.
For analysis by flow cytometry, 10⁵ cells were seeded 24 h prior to treatment in a 24-well plate. Samples were treated with 2.5 µM cy5-gp100-CPP for 1 h, then extensively washed with PBS and trypsinized, stained with live/dead fixable aqua (BioLegend), and analyzed by flow cytometry.
To image and quantify surface-associated peptide compared with internalized peptide, DC2.4 cells were incubated with 2.5 µM FITC-gp100-pAntp or FITC-gp100 for 4 h then scraped and washed with PBS to remove free-floating peptide. Cells were stained with live/dead fixable aqua (BioLegend) and analyzed by flow cytometry.

To evaluate the effects of the identified mutations on microtubule behavior, the transfected HeLa cells were fixed in 4% paraformaldehyde in PBS and blocked at room temperature for 1 h. Anti-FLAG-Cy3 antibody (1:500 dilution, Sigma-Aldrich) was used for determining the TUBA4A-FLAG localization, anti-β-tubulin antibody (1:500 dilution, Sigma-Aldrich) was used for detecting the endogenous microtubule network, and the DNA was labeled with Hoechst 33,342 solution (1:700 dilution, BD). The HeLa cells were observed and images were captured on a confocal laser-scanning microscope (LSM880, Zeiss) with a 63 × oil objective. For the quantification of microtubule phenotypes, approximately 200 cells expressing either wild-type or mutant TUBA4A were detected and classified according to the level of expression of the overexpressed plasmids (as judged by the intensity of the fluorescent label) in each of two independent experiments.

Curcumin, cycloheximide, and cisplatin were purchased from Sigma Aldrich (St. Louis, Missouri, United States). Antibodies against caspase-9, caspase-8, caspase3, cleaved caspase-3, PARP, XIAP, cIAP1, cIAP, Bcl2, Bclxl, Skp2, p27, p27, tubulin, ubiquitin, etc., were purchased from Cell Signaling Technologies (Beverly, MA, USA). GAPDH antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Annexin V-FITC, Propidium iodide staining solution, Hoechst 33342 Solution, BD Cytofix/Cytoperm plus fixation and permeabilization solution kit, BD MitoScreen (JC-1) Kit, were purchased from BD Biosciences (NJ, USA).

As described [26 (link)], NSCs were harvested 24 h after plasmid transfection or 48 h after lentivirus infection and then fixed with ice-cold 75% ethanol at -20°C for at least 24 h. Next, cells were stained with Hoechst 33342 solution (BD Biosciences) and incubated for 30 min at RT, washed, and suspended in BD Pharmingen™ Stain Buffer (BD Biosciences). The results were analyzed using the BD FACSCalibur system (BD Biosciences) with ModFit LT v3.3.11 software (Verity Software House).

Cells obtained after enzymatic digestion of testis were resuspended in 5–10 ml FACS buffer (1x DPBS [Gibco] supplemented with 1% Pen-Strep, 1% FBS, 10 mm HEPES, 1 mm pyruvate [Gibco] and 1 mg/ml glucose [Gibco]) and counted with a hemocytometer. Cells were diluted at 106/100 µl in FACS buffer. 1 µl of Hoechst 33342 Solution (BDPharmingen, stock: 1 mg/ml) and 0.1 µl of MitoTracker Deep Red (Invitrogen, stock: 1 mM) were added per 100 μl cell suspension, then cells were incubated at 35°C for 20 min. Thereafter, cells were kept on ice at all times. Cells were washed twice with ice-cold FACS buffer and the Sertoli cells fraction was sorted. Briefly, cell debris and doublets were gated out and remaining cells were gated for diploidy using the Hoechst channel. Diploid cells were further gated for high FSC and subsequently for a high signal in the MitoTracker channel.

Curcumin, cycloheximide, and cisplatin were purchased from Sigma Aldrich (St. Louis, Missouri, United States). Antibodies against caspase-9, caspase-8, caspase3, cleaved caspase-3, PARP, XIAP, cIAP1, cIAP, Bcl2, Bclxl, Skp2, p27, p27, tubulin, ubiquitin, etc., were purchased from Cell Signaling Technologies (Beverly, MA, USA). GAPDH antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Annexin V-FITC, Propidium iodide staining solution, Hoechst 33342 Solution, BD Cytofix/Cytoperm plus fixation and permeabilization solution kit, BD MitoScreen (JC-1) Kit, were purchased from BD Biosciences (NJ, USA).