Fig 1: Myeloid cells express SBSN in bone marrow of MDS patients. Immunofluorescence detection of CD11b (green) using anti-CD11b-BV421, SBSN (red) using anti-SBSN and Alexa Fluor 568 goat anti-rabbit in bone marrow smears of (A) MDS (EB-2) and (B) MDS 5q-syndrome patients. Nuclear DNA is stained by SiR-DNA (blue), with n = 3 per group. Scale bar, 10 µm. (C) Gating strategy used for isolation of eMDSCs, MDSCs, and monocyte populations from the bone marrow of MDS (n = 6) and ‘hematological malignancy’ patients (HM; multiple myelomas, T-cell lymphoproliferation, chronic anemia; n = 4). (D) SBSN mRNA levels of BM myeloid subpopulations eMDSCs [Lin- (CD3, 14, 16, 19, 20, 56) HLA-DR-, CD33+], PMN-/MN-MDSCs (MDSCs; Lin+, HLA-DR-, CD33+), monocytes (Lin+, HLA-DR+, CD33++), adipocytes, and granulocyte-containing high-density fraction in MDS (n = 4) and HM (multiple myelomas and T-cell lymphoproliferation; n = 3) estimated after cell sorting by RT–qPCR. (E) Comparison of the bone marrow subpopulation percentage in MDS (n = 6) and HM (multiple myelomas and chronic anemia; n = 3) patients. For patients' samples, one-way ANOVA was used for multigroup comparisons followed by the Tukey's post hoc test. Results are the means ± SD; *P < 0.05, **P < 0.01.
Fig 2: Polymorphonuclear and mononuclear cells express SBSN in bone marrow of MDS patients. (A) Immunofluorescence detection of SBSN (green) using anti-SBSN and Alexa Fluor 647 goat anti-rabbit in bone marrow smears of the MDS (EB-2; n = 3) and HM (Hodgkin's lymphoma; n = 3) groups. Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (B) Immunohistochemistry of SBSN in bone marrow smears of MDS patients using anti-SBSN antibody and IgG-HRP goat anti-rabbit (EB-1, and EB-2; n = 3; scale bar, 10 µm) and (C) quantification of SBSN-positive polymorphonuclear (PMN; 56%) and mononuclear (MN; 23%) cells in MDS patients' bone marrow smears. 100 PMN and 100 MN cells were counted from each sample (n = 3). Results are expressed as the means ± SD. (D) Immunohistochemistry of SBSN using anti-SBSN antibody and IgG-HRP goat anti-rabbit in bone marrow sections of the MDS group (MDS-MLD and 5q-; n = 3). Scale bars, 50 µm (larger image) and 10 µm (smaller image).
Fig 3: SBSN transcript is induced in vitro by IFN-? and 5-AC treatment and is present in MDS patients' bone marrow. RT–qPCR quantification of SBSN mRNA fold change in OCI-M2 and SKM-1 leukemic cell lines treated with (A) 5-AC (1 µm) and (B) IFN-? (5 ng·mL-1) for 72 h, with n = 3 per group. RT–qPCR quantification of SBSN mRNA fold change in (C) whole bone marrow samples of cohort #1 three groups of donors ‘MDS’ (n = 30), ‘HM’ (n = 19), and ‘Healthy’ (n = 8) and (D) in cohort #2 bone marrow mononuclear cell samples of two groups of donors ‘MDS’ (n = 48) and ‘HM’ (n = 11). (E) RT–qPCR quantification of SBSN mRNA fold change in whole bone marrow samples of newly diagnosed [‘5-AC(-)’, n = 20] and therapy-undergoing [‘5-AC(+)’, n = 10] patients. The two-tailed Mann–Whitney U-test was used for two-sample comparison. For patients' samples, one-way ANOVA was used for multigroup comparisons followed by the Tukey's post hoc test. The two-tailed unpaired t-test was used for two-group comparisons. Results are expressed as the means ± SD; *P < 0.05, **P < 0.01.
Fig 4: SBSN is a secreted protein present in MDS patients' bone marrow and peripheral blood. Anti-SBSN ELISA detection of SBSN protein levels (pg·mL-1) (A) in the conditioned medium of MCF-7 pLVX Tet-On empty/SBSN-1 cell lines and (B) in bone marrow plasma of cohort #3 four groups of patients ‘MDS’ (n = 42), ‘MDS 5q(-)’ (n = 12), ‘AML’ (n = 7), and ‘non-MDS’ (n = 12). Comparison of SBSN protein levels in BM of MDS patients prior (‘MDS’) and after (‘AML’) leukemic transformation (C). BM SBSN protein levels in MDS patients without 5-AC therapy [‘5-AC(-)’; n = 21] and patients undergoing 5-AC therapy [‘5-AC(+)’; n = 21] (D). Correlation between SBSN protein levels (pg·mL-1) (E) in bone marrow plasma and peripheral blood of MDS patients (n = 11) and (F) the mean comparison. The Kruskal–Wallis test was used for multiple comparisons followed by Dunn's multiple comparison test. The two-tailed paired t-test was used for statistics in panel C. The two-tailed Mann–Whitney U-test was used for two-sample comparison. Pearson's correlation coefficient was used for correlation statistics. Results are expressed as the means ± SD; *P < 0.05, **P < 0.01.
Fig 5: SBSN protein levels anticorrelate with MDS patients' bone marrow CCL2 chemokine levels and T lymphocyte count. (A) Correlation matrix of log2 SBSN protein levels (pg·mL-1) and TNF-a, IL-12, IL-1ß, IL-6, IP-10, IL-1a, IL-10, IL-27, IL-8, and CCL2 cytokine log2 levels (pg·mL-1), ***P < 0.001. (B) Correlation between log2 SBSN protein (pg·mL-1) and log2 CCL2 levels (pg·mL-1) in bone marrow of MDS, MDS 5q-, and AML patients (n = 52). Spearman's correlation coefficient was used for correlation statistics. (C) Correlation matrix of log2 SBSN protein levels (pg·mL-1) and T, B lymphocytes and blast percentages in BM, ***P < 0.001. (D) Correlation between log2 BM SBSN protein levels (pg·mL-1) and log2 BM T lymphocyte percentage of MDS, MDS 5q-, and AML patients (n = 53). (E) Correlation between SBSN mRNA fold changes of MDS patients' mononuclear cell samples and corresponding blast count in percentage. Spearman's correlation coefficient and Pearson's correlation coefficient were used for correlation statistics of non-normal data distribution (D) and normal data distribution (E), respectively. P-values were adjusted using the Benjamini–Hochberg false discovery rate method.
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