MicroRNAs Modulate the Noncanonical NF- B Pathway by Regulating IKK Expression During Macrophage Differentiation Tao Li 1 *, Michael J. Morgan 1 *, Swati Choksi 1, Yan Zhang 1, You-Sun Kim 2#, Zheng-gang Liu 1# 1 Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr., Bethesda, MD 20892 2 Institute for Medical Sciences, Ajou University School of Medicine, Suwon, 443-749 Korea *These authors contributed equally to this work and are listed alphabetically by their last names. # To whom correspondence should be addressed. Figure S1. Monocyte differentiation Mo Mo GM-CSF-7days Figure S1. Monocyte differentiation. The purity of the elutriated monocytes was checked by staining with anti-cd14 and analyzing by flow cytometry (upper panel). Monocytes (Mo) were differentiated into Macrophages (M )by culturing 7 days in presence of rhgm-csf, as shown by phase contrast microscopy (bottom panel).
Figure S2. Treatment of monocytes with GM-CSF alone leads to macrophage differentiation and not to dendritic cell differentiation CD 14 CD 1a GM-CSF CD 14 CD 1a GM-CSF + IL-4 CD 14 CD 1a GM-CSF + IL-6 Figure S2. Treatment of monocytes with GM-CSF alone leads to macrophage differentiation and not to dendritic cell differentiation. Monocytes were cultured for 7 days in presence of rhgm-csf alone (top). Differentiated cells were analyzed by FACS using antibodies to CD14 or CD1a (red, unstained; blue, stained.) For comparison, we induced dendritic cell fate by culturing in the presence of rhgm-csf plus IL-4. As a second comparison, we cultured in the presence of rhgm-csf and IL-6, which has been reported to restrict cell linage to the macrophage fate. No significant change in these markers was observed, suggesting that in our hands, rhgm-csf alone was sufficient to induce macrophage differentiation.
Figure S3. IKKα is up-regulated during monocyte differentiation #1 #2 #3 #4 Mo Mo Mo Mo - IKKα - TRADD Figure S3. IKK! is up-regulated during monocyte differentiation. Cell extracts of monocytes (Mo) vs. macrophages (M ) from different donors were analyzed by immunoblot with anti- IKK!, TRADD blots indicate the loading of lanes.
Figure S4. IKK! is downregulated by transfected mirna mimic pool during monocyte differentiation Monocyte GM-CSF: - + + - + - ctrl pool - - - IKKα - TRADD - β-actin Figure S4. IKKα is downregulated by transfected mirna mimic pool during monocyte differentiation. Monocytes (Mo) were transfected with an mirna mimic pool (mir-15a, mir16, mir) at day 0 and were analyzed after 4 days by immunoblot with anti- IKKα antibody. Actin and TRADD blots indicate the loading of lanes. Transfection of these cells by mirna or sirna was successful, as shown by western blot, which shows downregulation of IKK levels in the pool-transfected cells. However, control or pool- transfected cells failed to fully differentiate (as detected by morphology) into macrophages. As noted in the blot, the control cells did not upregulate IKK as highly as untransfected cells and died before 7 days of GMCSF treatment.
Figure S5. Down-regulation of IKKα in Hela cells by an mirna mimic pool results in less p52 processing mirna mimics HeLa Ctrl: + - 15a: - + 16: - + : - + - IKKα - p100 - p52 - TRADD - β-actin Figure S5. Down-regulation of IKK in Hela cells by an mirna mimic pool results in less p52 processing. HeLa cells were transfected with micrornas mimic control oligo and pooled mirna mimics 15a, 16, and. 48 hours after transfection, cell lysates were analyzed by immunoblot with anti- IKK, anti-p100/p52, TRADD and β-actin blots indicate loading of lanes.
Figure S6. Decreased p52 processing in HeLa cells transfected with the microrna mimic pool correlates not only with decreased IKKα levels, but also increased TRAF2 levels and decreased NIK levels. Hela - IKKα - TRADD - TRAF2 - NIK - TRADD Figure S6. Decreased p52 processing in HeLa cells transfected with the microrna mimic pool correlates not only with decreased IKKa levels, but also increased TRAF2 levels and decreased NIK levels. HeLa cells were transfected with micrornas mimic control oligo and pooled mirna mimics 15a, 16, and. 48 hours after transfection, cell lysates were analyzed by immunoblot with anti- IKK, anti-traf2, anti-nik antibodies. TRADD blots indicate loading of lanes in the two blots of the same lysate.
Figure S7. The noncanonical NF-κB pathway regulates basal ELC expression Relative Quantity 2.0 1.5 1.0 0.5 ELC mrna 0.0 Figure S7. The noncanonical NF- B pathway regulates basal ECL expression. Macrophages (M ) were transfected with mirna mimic control or pooled mirna mimics. 48 hours after transfection, total RNA was extracted and mrna of ELC were measured by real time PCR, ELC mrna level was normalized to GAPDH mrna, data representative of three independent experiments.
Figure S8. Canonical and noncanonical NF-κB pathways are capable of upregulating ELC expression. Relative Quantity 400 350 300 250 200 150 100 50 ELC mrna 0 TNFα: - + - + LTα 1 β 2 : - - + + Figure S8. Canonical and noncanonical NF- B pathways are capable of up-regulating ELC expression. Macrophages (M ) were pre-treated with LTα1β2 for 4 hours or not followed by 6 hours of TNFα treatment as indicated, total RNAs were isolated and ELC mrna was measured by real time PCR, ELC mrna level was normalized to GAPDH mrna, data are representative of three independent experiments.
Figure S9. mirna targeting IKKα affect basal canonical NF-κB gene expression. A20 mrna Basal ICAM mrna Basal Relative Quantity 1.25 1.00 0.75 0.50 0.25 Relative Quantity 1.50 1.25 1.00 0.75 0.50 0.25 0.00 0.00 Relative Quantity 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 CCL4 mrna Basal Relative Quantity 1.50 1.25 1.00 0.75 0.50 0.25 0.00 IL10 mrna Basal Figure S9. mirnas targeting IKK affect basal canonical NF- B gene expression Macrophages (M ) were transfected with mirnas control mimic or pooled mimics. 48 hours after transfection, total RNA was extracted and mrna of A20, ICAM, CCL4 and IL-10 were measured by real time PCR, target mrna level was normalized to GAPDH mrna, data are representative of three independent experiments.
Figure S10. p52-containing complexes have higher mobility than RelA or crel containing complexes, consistent with the formation of p52 homodimers. WCL P100 WCL P100 - BME: - - - - + + DSP: - + - + + + 250 KD- 150 KD- 100 KD- - p100 - p52 - p100 75 KD- 50 KD- - p52 WCL DSP: - + WCL DSP: - + 250 KD- 150 KD- 250 KD- - Rel A - c-rel 150 KD- 100 KD- 75 KD- 50 KD- - Rel A 100 KD- 75 KD- 50 KD- - c-rel Figure S10. p52-containing complexes have higher mobility than RelA- or crelcontaining complexes, consistent with the formation of p52 homodimers. Macrophages were cross-linked with DSP (1mM in PBS incubated at room temperature for 30 minutes) followed by p100 depletion by specific anti-p100 C-terminal antibody. The un-depleted and depleted cell lysated were analyzed by immunoblot with indicated antibodies. The cross-links were removed by mercaptoethanol (BME) in the right two lanes of the upper blot. Note that in crosslinked lanes RelA and crel are in higher molecular weight complexes than p52, as indicated by bands next to the arrows. (RelB in the unstimulated macrophages is present only at very low levels and was not detected in this experiment.) Since the Rel NF-kB proteins (RelA, RelB, c-rel), which contain transcriptional activation domains (TADs), are of higher molecular weight than the mature forms of nfkb1 (p50) and nfkb2 (p52), which do not contain TADs, this data suggests that p52 is substantially found in homodimers or heterodimers with p50, both of which would be transcriptionally inactive under normal conditions.
Figure S11. mirna targeting IKKα affect canonical NF-κB gene induction. Relative Fold Increase A20 mrna-lps Simulated 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Relative Fold Increase ICAM mrna-lps Simulated 6 5 4 3 2 1 0 Relative Fold Increase CCL4 mrna-lps Simulated 20 18 16 14 12 10 8 6 4 2 0 Relative Fold Increase IL-10 mrna-lps Simulated 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure S11. mirna targeting IKK affect canonical NF- B gene induction. Macrophages (M ) were transfected with mirnas control mimic or pooled mirna mimics. 24 hours after transfection. cells were challenged or not with LPS (1 g/ml) for another 24 hours, total RNA were isolated and mrnas of A20, ICAM, CCL4 and IL-10 were measured by real time PCR, target mrna levels were normalized to GAPDH mrna, fold increase compare with the untreated cells is shown, data representative of three independent experiments.
Figure S12. sirna targeting IKKα or p52 affect noncanonical NF-κB gene expression and induction. a 2.5 ELC mrna Basal b 4 ELC mrna Basal 2.0 3 1.5 2 1.0 0.5 1 0.0 sirna: Lamin A/C NT IKKα Mo 0 sirna: Lamin A/C p52 c sirnas: - NT p52 - p100 - p52 - TRADD - β-actin d 800 ELC mrna Induced e 12.5 A20 mrna Induced Relative Fold Increase 600 400 200 0 NT IKKα Relative Fold Increase 10.0 7.5 5.0 2.5 0.0 NT IKKα Figure S12. sirna targeting IKK or p52 affect noncanonical NF- B gene expression and induction. (a,b,c,d,e) Macrophages (M ) were transfected with Nontargeting (NT) control, lamin A/C, p52/p100 or IKK sirna as indicated. 24 hours after transfection. cells were challenged or not with LPS (1 g/ml) for another 24 hours. For various experiments, total RNA were isolated and mrnas were measured by real time PCR with target mrna levels normalized to GAPDH mrna. (c) Lysates from monocytes (Mo) and sirna-transfected macrophages (M ) were analyzed by immunoblot with indicated antibodies. (d,e) Fold increase compared with the untreated cells is shown instead of relative levels.