![S100A8 and S100A9 is induced via IL-6/Jak/STAT3 signaling. (A) CM from HS-5 cells (n = 8) was analyzed with a bead-based multiplex assay for 25 human cytokines (IL-9, IL-10, IL-17A, and interferon α [IFN-α] not detected). The pie chart displays the individual proportion among the total analytes. (B) Induction of S100A8 and S100A9 on the mRNA (left; OCI, n = 4; MOLM-13, n = 5) and induction of an S100A8/A9hi population (right; OCI-AML, n = 4; MOLM-13, n = 4) were analyzed after treatment with 10 ng/mL of rhIL-6 for 48 hours by qPCR and flow cytometry, respectively. mRNA levels are shown as the fold change with or without rhIL-6 (untreated set as 1). (C) Frequency of S100A8/A9hi AML cells was determined by flow cytometry after culturing HS-5 CM-treated MOLM-13 cells (n = 4) in the absence (isotype) or presence of 10 μg/mL anti-IL-6 or anti-IL-6R blocking antibodies. (D) CM from HS-5 cells was prepared after inflammatory priming of the HS-5 cells (with 15 ng/mL tumor necrosis factor-α and 10 ng/mL IFN-γ). Gene expression of IL-6 in HS-5 cells was determined by qPCR and expressed as fold change between primed and nonprimed samples (nonprimed samples set as 1; left, n = 4). The frequency of S100A8/A9hi AML cells (right; OCI-AML, n = 2; MOLM-13, n = 4) cultured with nonprimed and primed HS-5 CM for 48 hours was determined by intracellular flow cytometry. (E) Gene expression of IL-6 was determined in healthy donor– and AML patient–derived BM-MSCs (both n = 5). (F) Gene expression of PIM1 and SOCS3 (2 STAT3 target genes) was determined in AML cell lines (OCI-AML n = 3, MOLM-13 n = 3) cultured in the absence or presence of HS-5 CM for 48 hours by qPCR and is shown as the fold change (untreated set as 1). (G) Phosphorylation of the STAT proteins (p-STAT) 1, 3, 5, and 6 was determined by PhosFlow in AML cell lines (OCI-AML, n = 3; MOLM-13, n = 3) cultured in the absence or presence of HS-5 CM for 20 minutes and is depicted as a heat map showing the fold change with or without HS-5 CM (left; untreated set as 1). Most prominently, p-STAT3 was induced by HS-5 CM treatment as representatively shown in the histogram (right; scale indicates median fluorescence intensity). (H-J) STAT3 phosphorylation was analyzed by PhosFlow in AML cell lines cultured in the presence of HS-5 CM for 20 minutes after pretreatment for 2 hours with anti-IL-6–blocking antibodies (H; OCI-AML, n = 3; MOLM-13, n = 3), ruxolitinib (I; OCI-AML, n = 2; MOLM-13, n = 2), and Jak1 or Jak2 inhibitor (J; OCI-AML, n = 2; MOLM-13, n = 2). (K-L) Induction of S100A8 and S100A9 gene expression after 48 hours of culture with HS-5 CM was analyzed in the absence (0 μM) or presence (1 μM) of the pan-Jak inhibitor ruxolitinib (K; OCI-AML, n = 3; MOLM-13, n = 3) or the p-STAT3 inhibitor C188-9 (L; OCI-AML, n = 5; MOLM-13, n = 5). Values are depicted as fold change with or without HS-5 CM (untreated set as 1). Data are expressed as the standard error of mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ns, not significant. qPCR, quantitative real-time polymerase chain reaction.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/6/21/10.1182_bloodadvances.2021005938/7/blooda_adv-2021-005938-gr2.jpeg?Expires=1719027051&Signature=DJRakBxoUYcvEUrE82J-7BpTG7B2vr1k0JyAvyHtE83CIvvqw6sQSgcP0tlkoe5MJapWZwJB49QJeAXji6yVXnbnQfBfOsys99qXuG3r2ZwL~5WWfA-87ElJ2ZpC2eNdpoBOdxkUIpmT1QFYaIdrw1JE1Ngc4-FUbj1blSpuGNGMzD0Xk5Wl4iwDCVRQI20bUnk25qOJfqZWBwxqXZpGYDIc7NyYTAnMoce~G61TrHa9p4DM6wkqwHxOJP3FEmmvZiu4By5ziRHoIlCqdx14hEsyptIbBCXth1fzYd9gcMHS~5CbOKsQGGNUtMUNpOU18m8EnZFVt0ffzjIA~G6HXQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
S100A8 and S100A9 is induced via IL-6/Jak/STAT3 signaling. (A) CM from HS-5 cells (n = 8) was analyzed with a bead-based multiplex assay for 25 human cytokines (IL-9, IL-10, IL-17A, and interferon α [IFN-α] not detected). The pie chart displays the individual proportion among the total analytes. (B) Induction of S100A8 and S100A9 on the mRNA (left; OCI, n = 4; MOLM-13, n = 5) and induction of an S100A8/A9hi population (right; OCI-AML, n = 4; MOLM-13, n = 4) were analyzed after treatment with 10 ng/mL of rhIL-6 for 48 hours by qPCR and flow cytometry, respectively. mRNA levels are shown as the fold change with or without rhIL-6 (untreated set as 1). (C) Frequency of S100A8/A9hi AML cells was determined by flow cytometry after culturing HS-5 CM-treated MOLM-13 cells (n = 4) in the absence (isotype) or presence of 10 μg/mL anti-IL-6 or anti-IL-6R blocking antibodies. (D) CM from HS-5 cells was prepared after inflammatory priming of the HS-5 cells (with 15 ng/mL tumor necrosis factor-α and 10 ng/mL IFN-γ). Gene expression of IL-6 in HS-5 cells was determined by qPCR and expressed as fold change between primed and nonprimed samples (nonprimed samples set as 1; left, n = 4). The frequency of S100A8/A9hi AML cells (right; OCI-AML, n = 2; MOLM-13, n = 4) cultured with nonprimed and primed HS-5 CM for 48 hours was determined by intracellular flow cytometry. (E) Gene expression of IL-6 was determined in healthy donor– and AML patient–derived BM-MSCs (both n = 5). (F) Gene expression of PIM1 and SOCS3 (2 STAT3 target genes) was determined in AML cell lines (OCI-AML n = 3, MOLM-13 n = 3) cultured in the absence or presence of HS-5 CM for 48 hours by qPCR and is shown as the fold change (untreated set as 1). (G) Phosphorylation of the STAT proteins (p-STAT) 1, 3, 5, and 6 was determined by PhosFlow in AML cell lines (OCI-AML, n = 3; MOLM-13, n = 3) cultured in the absence or presence of HS-5 CM for 20 minutes and is depicted as a heat map showing the fold change with or without HS-5 CM (left; untreated set as 1). Most prominently, p-STAT3 was induced by HS-5 CM treatment as representatively shown in the histogram (right; scale indicates median fluorescence intensity). (H-J) STAT3 phosphorylation was analyzed by PhosFlow in AML cell lines cultured in the presence of HS-5 CM for 20 minutes after pretreatment for 2 hours with anti-IL-6–blocking antibodies (H; OCI-AML, n = 3; MOLM-13, n = 3), ruxolitinib (I; OCI-AML, n = 2; MOLM-13, n = 2), and Jak1 or Jak2 inhibitor (J; OCI-AML, n = 2; MOLM-13, n = 2). (K-L) Induction of S100A8 and S100A9 gene expression after 48 hours of culture with HS-5 CM was analyzed in the absence (0 μM) or presence (1 μM) of the pan-Jak inhibitor ruxolitinib (K; OCI-AML, n = 3; MOLM-13, n = 3) or the p-STAT3 inhibitor C188-9 (L; OCI-AML, n = 5; MOLM-13, n = 5). Values are depicted as fold change with or without HS-5 CM (untreated set as 1). Data are expressed as the standard error of mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ns, not significant. qPCR, quantitative real-time polymerase chain reaction.
