Fig 1.
Workflow for preparing cell samples for proteomic studies.
Fig 2.
The impact of HL-60 cell proliferation by DMSO or DMF treatment.
HL-60 cells were differentiated to neutrophil-like cells by DMSO or DMF. HL-60 cells (2 × 10⁵/mL) were seeded into a T-75 flask and treated with 1.25% DMSO or 70 mM DMF by addition of fresh agent on days 0, 1, and 3 for a total differentiation period of five days Cell viability was assessed using the trypan blue dye exclusion assay. (*, **, and ***) indicates comparison with untreated HL-60 cells (* P < 0.05, **P ≤ 0.01, *** P < 0.0001). (#) indicates comparison between DMSO-df- and DMF-df-HL-60 cells at the same time point (# P < 0.05). ($) indicates comparison between Day 3 and Day 5 within the same group ($ P < 0.05). The growth curve showed rapid growth in untreated HL-60 cells compared to differentiated cells following DMSO and DMF treatments. Both DMSO and DMF showed no significant proliferation after day 3, with a significant decrease in DMSO-df-HL-60 cells. Data are presented as mean ± SD of viable cells (n = 3).
Fig 3.
Quantification of CD11b expression in untreated and differentiated HL-60 cells.
(A) Flow cytometry gating strategy: Cells were first gated based on FSC/SSC profiles to identify the main cell population, followed by gating for single cells and CD11b stained cells. (B) The histograms show percentages of CD11b-positive cells. DMSO-df-HL-60 cells demonstrated the highest expression of CD11b compared to DMF-df-HL-60 cells and untreated cells. (C) Bar graph summarizing the percentage of CD11b-positive cells across the three groups. Data are represented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA with significance defined as: (****P < 0.0001, *P < 0.05).
Fig 4.
Measurement of superoxide anion production in undifferentiated and differentiated HL-60 cells.
The absorbance units represent the relative levels of ROS production. $ indicates comparison of PMA-stimulated DMSO- or DMF-df-HL-60 cells with PMA-stimulated undifferentiated HL-60 cells. * indicates comparison between between each PMA-stimulated group and its corresponding untreated group. The bars represent the mean ± SD of three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA with significance defined as follows: (**P < 0.01, ****P < 0.0001, $$$$ P< 0.0001).
Fig 5.
Identification of superoxide anion radical (O₂•⁻) from differentiated HL-60 cells.
The reactions were performed using untreated HL-60, DMSO-, and DMF-df-HL-60 cells. Cells were treated with 100 mM DMPO in 100 µL HBSS and stimulated with 1 µM PMA to induce the respiratory burst. The spectra were obtained from 1 × 10⁶ cells after 15 minutes of gentle mixing (300 rpm) at 37 °C for untreated HL-60 (A), DMSO-df-HL-60 (B), and DMF-df-HL-60 (C) cells. Hyperfine splittings corresponding to superoxide (DMPO/•OOH) (marked by stars) and DMPO/•OH (marked by dots) were identified and shown on the simulated spectrum (D). The reaction mixtures were transferred to 3 x 50 µL micro-capillary tubes and placed in a 3 mm EPR tube for spectral acquisition. In D, the simulated spectrum (correlation of r = 0.97) of DMF-df-HL-60 cells (in C) is shown. It is a composite spectrum of DMPO/•OH (hyperfine splitting constants: aN = aH = 14.9 G) and DMPO/•OOH (hyperfine splitting constants: aN = 14.2 G, aHβ = 11.1 G, aHγ = 1.3 G).
Fig 6.
Myeloperoxidase (MPO) activity in untreated HL-60, DMSO-df-HL-60, and DMF-df-HL-60 cells.
The 1-minute time point was selected for comparison among the groups to represent MPO activity. The results are expressed as a percentage, with undifferentiated HL-60 cells set as the reference (100%). Statistical analysis was performed using one-way ANOVA. The data show that MPO activity significantly decreased in both DMSO- and DMF-df-HL-60 groups by around 50% compared to undifferentiated HL-60 cells (**P < 0.01). The bars represent the mean ± SD of three independent experiments (n = 3).
Fig 7.
Differentiated HL-60 cells reveal a stepwise decrease in MPO expression over five days.
MPO expression in HL-60 cells differentiated with DMSO (left) or DMF (right) over a five-day period. Untreated HL-60 cells were used as the baseline control, while the 0-day treated group refers to cells treated with either DMSO or DMF for 15 minutes before lysis. Data were expressed as mean ± SD, with triplicate experiments (n = 3).). Statistical analysis was performed using one-way ANOVA with significance defined as(*P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001).
Fig 8.
Comparison of NET formation in DMSO- or DMF-differentiated HL-60 cells.
(A) NET formation was quantified using a Sytox Green plate reader assay after differentiation by DMSO or DMF. Differentiated HL-60 cells were left unstimulated (control) or stimulated with PMA (500 nM) (NOX-dependent) or A23187 (4 µM) (NOX-independent) for 6 hour. Where indicated, cells were pre-treated with diphenyleneiodonium (DPI, 20 µM) for 1 hour prior to stimulation. NETosis was reported as Sytox Green fluorescence (RFU). Data are presented as mean ± SEM from three independent experiments (n = 3).. Statistical analysis was performed using one-way ANOVA with significance defined as (*P < 0.05 and ****P < 0.0001). (B) Representative fluorescence microscopy images showing NET formation in DMSO- or DMF-differentiated HL-60 cells. Cells were left untreated (control) or stimulated with PMA (500 nM) for 6 hour, fixed, and stained for MPO (FITC) and DNA (Sytox Red). Scale bar: 50 µm, representative image (n = 3).
Fig 9.
Visualization of protein expression reveals proteomic similarity between DMSO- and DMF-treated cells.
A three-way tri-plot was used to visualize the differences in protein abundance across control, DMSO, and DMF -treated samples. The log2 ratio of normalized protein abundance was plotted to highlight the variations between the three groups. In this visualization, the proteins with higher abundance in one group relative to the others are positioned closer to the corresponding vertex. Significantly abundant proteins (P < 0.05) are shown by yellow circles.
Fig 10.
Enriched gene ontology pathways are based on the proteomic analysis of differentiated HL-60 cells treated with DMSO or DMF.
Gene ontology analysis was performed on unique proteins from each group. The number of proteins from gene ontology are shown in dotted circles. A detailed list of proteins and gene ontology analysis are provided in S1 Table..
Fig 11.
Heatmap of the genes associated with neutrophil degranulation during HL-60 differentiation.
The differential expression of genes across untreated HL-60 cells (C1-C5), differentiated cells treated with DMF (DMF1-DMF5) and DMSO (DMSO1-DMSO5). Yellow indicates upregulated proteins, while blue indicates downregulated proteins.
Fig 12.
Proteomes of DMF- and DMSO-df-HL-60 cells.
Protein abundances are represented by polygon size and grouped by functional categories. A: Broad functional categories reflect the overall functional organization of the proteome. B: Pathways involved in cellular adaptations that support differentiation. C: Enriched pathways critical for cellular functions and processes. D: Highly abundant genes associated with key cellular activities.
Fig 13.
Protein abundance for neutrophil-like function markers in the differentiated HL-60 cells: A: S100A9 protein abundance, associated with key neutrophil functions, was significantly upregulated in differentiated HL-60 cells, with higher expression in DMF-treated cells compared to DMSO-treated cells.
B: MPO protein amount, which decreases during differentiation, was reduced in both DMSO- and DMF-treated cells. C: FPR1 protein abundance, a late marker of granulocyte differentiation, significantly increased in HL-60 cells differentiated with DMSO or DMF compared to undifferentiated HL-60 cells. The protein expression levels were higher in DMSO-treated cells. D: The amount of CD11b protein, an early marker of neutrophil differentiation, significantly increased in HL-60 cells differentiated with DMSO or DMF compared to undifferentiated HL-60 cells. The protein expression levels were higher in DMSO-treated cells. Data are presented as mean ± SD from five independent experiments (n = 5). Statistical analysis was performed using one-way ANOVA with significance. (**P < 0.01 ***P < 0.001, ****P < 0.0001).