To analyze septation and subcellular localization, fresh conidia and mycelia of the Fon strains were washed with sterile ddH2O2 and co-stained with CFW or Hoechst 33342, respectively [9 (link)]. For apoptotic cell death analysis, the germ tubes of the Fon strains were treated with/without apoptosis-inducing compound farnesol (FOH; Shanghai Aladdin Bio-Chem, Shanghai, China) at 25 or 50 μM for 4 h at 26 °C. Necrotic cells and nuclei were co-stained with propidium iodide (PI; Shanghai Yeasen Biotech, Shanghai, China) and Hoechst 33342 (Shanghai Yeasen Biotech, China), respectively [35 (link),36 (link)]. To examine autophagy, the Fon strains were cultivated in liquid CM for 12 h, and the collected mycelia were rinsed with sterile ddH2O2. After washing, the mycelia were transferred into nitrogen-starved MM medium (MM-N) with/without 4 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO, USA) for 1 h. The mycelia were stained with the fluorescent dye monodansylcadaverine (MDC; Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark, followed by ddH2O2 washing [37 (link),38 (link)]. All aforementioned samples were observed under a Zeiss LSM 780 Meta confocal microscope (Gottingen, Niedersachsen, Germany) with the appropriate excitation and emission filters for corresponding dyes and signals.
Hoechst 33342
Manufactured by Yeasen
Sourced in China, Germany
Hoechst 33342 is a fluorescent DNA-binding dye used for staining and visualizing nuclei in live and fixed cells. It binds to the minor groove of double-stranded DNA and emits blue fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Yeasen (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Triton x 100, by Merck Group (2 mentions)
Metaxpress software, by Molecular Devices (2 mentions)
Imagexpress micro confocal system, by Molecular Devices (2 mentions)
Calcein am, by Yeasen (2 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (2 mentions)
Rpmi 1640 medium, by Yeasen (2 mentions)
Chlorin e6 ce6, by J&K Scientific (2 mentions)
Dialysis membrane 2 kd, by Solarbio (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Ros assay kit, by Beyotime (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Monodansylcadaverine mdc, by Merck Group (1 mentions)
Lsm 780 meta confocal microscope, by Zeiss (1 mentions)
Phenylmethanesulfonyl fluoride pmsf, by Merck Group (1 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (1 mentions)
38 protocols using hoechst 33342
1
Subcellular Localization and Apoptosis Assay for Fon
2
Immunofluorescence Analysis of Autophagy Markers
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Paraformaldehyde-fixed, Triton X-100-permeabilized NRVMs were subjected to immunofluorescence staining to analyze the expression and localization of VEGF-B, GRP78 and ATF4. Hoechst 33342 (Yeasen Biotech, China) and 6-diamino-2-phenylindole (DAPI) were used to nuclear staining (St. Louis, MO, USA). Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, NY, USA) was used to detect the autophagic flux, according to the manufacturer’s protocol. The confocal microscope (Olympus FV1000) was used for capturing images with high resolutions.
3
Immunofluorescence Assay for DNA Damage and Proliferation
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To detect DNA damage and the proliferative activity of cell γ-H2AX, Ki67 immunofluorescence staining was performed. Specifically, cells were fixed with 4% paraformaldehyde and exposed to 0.25% Triton X-100 (Sigma, USA) in PBS solution for 10 min at room temperature. The cells were then blocked with 5% BSA solution for 30 min and incubated with the primary antibody anti-γH2AX or anti-Ki67 (1 : 100, Abcam) overnight at 4°C. After washing the cells with PBS, the cells were further incubated with a fluorescently tagged secondary antibody (1 : 500, Abcam) for 1 h at room temperature in the dark. Subsequently, Hoechst 33342 (20 μg/ml, Yeasen, China) was used to stain the nuclei. Finally, immunofluorescence staining was observed under a fluorescence microscope (Leica DMi8, Germany).
4
Hoechst 33342 Nucleus Staining
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The nucleus morphology was dyed using Hoechst 33342 (Yeasen, Shanghai, China) according to the manufacturer’s instructions and was observed with a fluorescence microscope (Olympus Inc., Tokyo, Japan).
5
Mitochondrial ROS Measurement in HUVECs
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Mitochondrial ROS in HUVECs was measured by MitoSOX Red Mitochondrial Superoxide Indicator (Yeasen, #40778ES50, China) according to manufacturer’s instruction. For nuclei staining, Hoechst 33,342 (Yeasen, #40732ES03, China) was used at 5 μg/ml. And then images of fluorescent were observed and collected by Confocal laser-scanning microscopy (Nikon).
6
Autophagy Visualization in HepG2 Cells
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HepG2 and HepG2/CDDP cells were plated on 96-well plates and reached 60% -70% confluence at the time of transfection. The cells were transfected with Ad-mCherry-GFP-LC3B adenovirus (Hanbio Biotech, Shanghai, China) at an MOI of 30 in 200 µl of DMEM medium containing 10% FBS for 6 h at 37 °C. Following treatment with calyxin Y and/or CDDP for 18 h, the cells were stained with Hoechst 33342 (Yeasen). The stained cells were washed with PBS twice, observed with an ImageXpress® Micro Confocal system (Molecular Devices), and analyzed with MetaXpress software (Molecular Devices).
7
Calyxin Y and CDDP Cytotoxicity Assay
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HepG2 and HepG2/CDDP cells were plated on 96-well plates and incubated overnight. Next, the cells were treated with calyxin Y and/or CDDP for 24 h. Then, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% BSA. To detect AIF, cytochrome C and LC3B, the cells were incubated with indicated primary antibodies overnight at 4 °C. Then, the cells were incubated with an Alexa Flour 488-conjugated secondary antibody for 2 h. The mitochondria were stained with MitoTracker® Deep Red FM (Invitrogen). The nuclei were stained with Hoechst 33342 (Yeasen). The stained cells were washed with PBS twice, observed with an ImageXpress® Micro Confocal system (Molecular Devices), and analyzed with MetaXpress software (Molecular Devices).
8
Immunofluorescence Staining of Cells
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Glass coverslips was put into 6-well plate, and 3 × 105 cells were seeded on coverslips. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized with 0.3% Triton-X 100 and then blocked with 3% BSA for 1 h at room temperature. Post blocking, cells were incubated with the anti-Flag primary antibody (Yeasen, Shanghai, China, 30503ES20) overnight at 4 °C, then washed with PBS at room temperature and incubated with secondary antibody conjugated to Alexa® Fluor 594 (Yeasen, Shanghai, China, 33212ES60) for 1 h at room temperature. The cells were washed and stained using Hoechst 33,342 (Yeasen, Shanghai, China, 40731ES10) to visualize the nuclei. A laser scanning confocal microscope (Leica, Wetzlar, Germany, TCS-SP8) was also used to investigate the colocalization of EGFP and αEGFP TRIMbody; this instrument is equipped with a 405 nm violet laser, a 488 nm blue laser, a 561 nm green laser, and a 639 nm red laser.
9
Anticancer Potentials of TmSm Proteins
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E. coli BL21 (DE3), E. coli DH5α, pET-24a (+) were provided by Invitrogen (CA, USA). pET-24a (+)-TmSm34, pET-24a(+)-TmSm48, and pET-24a(+)-TmSm84 were previously constructed by our laboratory. MTT were supplied by Solarbio (Beijing, China). Hoechst 33342 were purchased from Yeasen (Shanghai, China). All other reagents were of analytical grade and acquired from Sigma-Aldrich (Shanghai, China). Human lung cancer cell A549 and normal liver cell L-02 was obtained from the Type Culture Collection Committee of Chinese Academy of Science (Shanghai, China). Fetal bovine serum (FBS), penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin), Roswell Park Memorial Institute 1640 medium (RPMI1640), Dulbecco’s Modified Eagle medium (DMEM), DMEM/Nutrient Mixture F-12 medium (DMEM/F12), and B27 were purchased from Gibco (Waltham, USA). Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were acquired from Pepcech (Cathy, USA).
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E. coli BL21 (DE3), E. coli DH5α, pET-24a (+) were provided by Invitrogen (CA, USA). pET-24a (+)-TmSm34, pET-24a(+)-TmSm48, and pET-24a(+)-TmSm84 were previously constructed by our laboratory. MTT were supplied by Solarbio (Beijing, China). Hoechst 33342 were purchased from Yeasen (Shanghai, China). All other reagents were of analytical grade and acquired from Sigma-Aldrich (Shanghai, China). Human lung cancer cell A549 and normal liver cell L-02 was obtained from the Type Culture Collection Committee of Chinese Academy of Science (Shanghai, China). Fetal bovine serum (FBS), penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin), Roswell Park Memorial Institute 1640 medium (RPMI1640), Dulbecco’s Modified Eagle medium (DMEM), DMEM/Nutrient Mixture F-12 medium (DMEM/F12), and B27 were purchased from Gibco (Waltham, USA). Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were acquired from Pepcech (Cathy, USA).
10
Multimodal Cytotoxicity Assay Protocol
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MTT, calcein-AM, EthD-1, AnnexinV-FITC/PI, ER-tracker, Mito-Tracker, Fluo-4-AM,
DAPI, Hoechst 33342, H2DCF-DA, MEQ, and BCECF-AM were purchased from Yeasen
(Shanghai, China). Z-VAD-fmk, 3-methyladenine (3-MA), bafilomycinA1 (Baf),
cycloheximide (CHX), N-acetylcysteine (NAC), reduced
glutathione (GSH), SB203580, U0126, SP600125, ruthenium red (RR), and disodium
4,4′-diisothiocyanato-2,2′-stilbenedisulfonate hydrate (DIDS) were obtained from
Selleck Chemicals (Houston, TX). The following antibodies were used: Cleaved
Caspase Antibody Sampler Kit 9929T (cleaved caspase-3, -7, and -9 and cleaved
poly-ADP-ribose polymerase [PARP]), Procaspase Antibody Sampler Kit 12742T
(Caspase-3, -7, and -9; PARP), Bcl-2 (3498T), Bax (2772T), Beclin-1(3495T),
LC3I/II (12741T), p62(5114S), ER Stress Antibody Sampler Kit 9956T (calnexin,
BiP, IRE1α, CHOP), ubiquitin (43124S), XBP1s (83418S), MAPK Family Antibody
Sampler Kit 9926T (ERK 1/2, p38, JNK), Phospho-MAPK Family Antibody Sampler Kit
9910T (phospho-ERK1/2, phospho-p38, phospho-JNK, Rabbit IgG HRP, Mouse IgG HRP),
GAPDH (5174S), and chloride intracellular channel-1 (CLIC1; 53424S) were
purchased from Cell Signaling Technology (Danvers, MA). Alexa Fluor 647
AffiniPure Goat anti-Rabbit immunoglobulin G (IgG; H + L) (cat: FMS-RBaf64701)
was obtained from FCMRCS (Nanjing, China).
DAPI, Hoechst 33342, H2DCF-DA, MEQ, and BCECF-AM were purchased from Yeasen
(Shanghai, China). Z-VAD-fmk, 3-methyladenine (3-MA), bafilomycinA1 (Baf),
cycloheximide (CHX), N-acetylcysteine (NAC), reduced
glutathione (GSH), SB203580, U0126, SP600125, ruthenium red (RR), and disodium
4,4′-diisothiocyanato-2,2′-stilbenedisulfonate hydrate (DIDS) were obtained from
Selleck Chemicals (Houston, TX). The following antibodies were used: Cleaved
Caspase Antibody Sampler Kit 9929T (cleaved caspase-3, -7, and -9 and cleaved
poly-ADP-ribose polymerase [PARP]), Procaspase Antibody Sampler Kit 12742T
(Caspase-3, -7, and -9; PARP), Bcl-2 (3498T), Bax (2772T), Beclin-1(3495T),
LC3I/II (12741T), p62(5114S), ER Stress Antibody Sampler Kit 9956T (calnexin,
BiP, IRE1α, CHOP), ubiquitin (43124S), XBP1s (83418S), MAPK Family Antibody
Sampler Kit 9926T (ERK 1/2, p38, JNK), Phospho-MAPK Family Antibody Sampler Kit
9910T (phospho-ERK1/2, phospho-p38, phospho-JNK, Rabbit IgG HRP, Mouse IgG HRP),
GAPDH (5174S), and chloride intracellular channel-1 (CLIC1; 53424S) were
purchased from Cell Signaling Technology (Danvers, MA). Alexa Fluor 647
AffiniPure Goat anti-Rabbit immunoglobulin G (IgG; H + L) (cat: FMS-RBaf64701)
was obtained from FCMRCS (Nanjing, China).
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