micrornas modulate the noncanonical nf- b pathway by ...micrornas modulate the noncanonical nf- b...

Figure S1. Monocyte differentiation

GM-CSF-7daysMo MФ

Mo

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).

MicroRNAs Modulate the Noncanonical NF- B Pathway by Regulating IKK Expression During Macrophage Differentiation

Tao Li1*, Michael J. Morgan1*, Swati Choksi1, Yan Zhang1, You-Sun Kim2#, Zheng-gang Liu1#

1Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr., Bethesda, MD 20892 2Institute 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.

Nature Immunology: doi:10.1038/ni.1918

Figure S2. Treatment of monocytes with GM-CSF alone leads tomacrophage differentiation and not to dendritic cell differentiation

CD 14 CD 1a

GM-CSF

GM-CSF + IL-4

GM-CSF + IL-6

CD 1a

CD 1a

CD 14

CD 14

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 .


- IKKα

- TRADD

Mo MФ Mo MФ Mo MФ Mo MФ

#1 #2 #3 #4

Figure S3. IKKα is up-regulated during monocyte differentiation

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.


Monocyte

miRNA mimics: - ctrl pool - - GM-CSF: - + + - +

- 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, miR223) 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 S4. IKK! is downregulated by transfected miRNA mimic pool during

monocyte differentiation


HeLam

iRN

A m

imic

sCtrl: + -15a: - +16: - +

223: - +

- IKKα

- β-Actin

- p100

- p52

Figure S5. Down-regulation of IKKα in Hela cells by an miRNA mimic poolresults in less p52 processing

- TRADD

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 223. 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 themicroRNA mimic pool correlates not only with decreased IKKα levels, butalso increased TRAF2 levels and decreased NIK levels.

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 223. 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.

Hela

miRNA mimics: 15a,16, 223Control

- NIK

- TRAF2

- TRADD

- TRADD

- IKKα


0.0

0.5

1.0

1.5

2.0

Rel

ativ

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uant

ity

ELC mRNA

MФ

Figure S7. The noncanonical NF-κB pathway regulates basal ELC expression

ControlmiRNA mimics: 15a,16, 223

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.


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ativ

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ELC mRNA

LTα1β2: - - + +TNFα: - + - +

MФ

Figure S8. Canonical and noncanonical NF-κB pathways are capable of up-regulating ELC expression.

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.


Rel

ativ

e Q

uant

ity

A20 mRNA Basal

Figure S9. miRNA targeting IKKα affect basal canonical NF-κB gene expression.

ICAM mRNA Basal

CCL4 mRNA Basal IL10 mRNA Basal

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1.00

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1.50

ControlmiRNA mimics: 15a,16, 223 ControlmiRNA mimics: 15a,16,

223

Control 15a,16, 223

Control 15a,16, 223miRNA mimics: miRNA mimics:

MФMФ

MФ MФ

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. 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 S10. p52-containing complexes have higher mobility than RelA or cRelcontaining complexes, consistent with the formation of p52 homodimers.

WCL

DSP: - + - + + +BME: - - - - + +

P100- WC

L

P100

- p52

- p52- p100

- p100

WCLDSP: - +

WCL

- Rel A

- Rel A - c-Rel

- c-Rel

DSP: - +

250 KD-

150 KD-

100 KD-

75 KD-

50 KD-

250 KD-

150 KD-

100 KD-

75 KD-

50 KD-

250 KD-

150 KD-

100 KD-

75 KD-

50 KD-


Rel

ativ

e Fo

ld In

crea

se

ICAM mRNA-LPS SimulatedA20 mRNA-LPS Simulated

CCL4 mRNA-LPS Simulated IL-10 mRNA-LPS Simulated

Rel

ativ

e Fo

ld In

crea

se

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ativ

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ld In

crea

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ControlmiRNA mimics: 15a,16, 223 ControlmiRNA mimics:

15a,16, 223

Control 15a,16, 223

Control 15a,16, 223miRNA mimics: miRNA mimics:

MФMФ

MФ MФ

Figure S11. miRNA targeting IKKα affect canonical NF-κB gene induction.

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.


siRNAs: - NT p52

Mo MФ

- β-Actin

- TRADD- p52

- p100

Figure S12. siRNA targeting IKKα or p52 affect noncanonical NF-κB gene expression and induction.

LaminA/C

Control

0

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600

800

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2.5ELC mRNA Basal

ELC mRNA Induced A20 mRNA Induced

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ld In

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ld In

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NTControlsiRNA: IKKα

0

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4ELC mRNA Basal

LaminA/C

Control

p52siRNA:

NTControl IKKα IKKα

0.0

2.5

5.0

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10.0

12.5

NTControl

b

c

a

ed

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.


micrornas modulate the noncanonical nf- b pathway by ...micrornas modulate the noncanonical nf- b...

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