GFP silencing reverted cells

X

GFP silenced cells

Vectors employed

GFP expressing cells

Sf21 cell

Figure 1A

Experimental line-1

Experimental line-2

Control line

a.

b.

c.

f.

1 2 3

1 2

i

ii

d. 1 2 3

21 nt

1 M 2

14 kDa

gfp shRNA (49 bp)EcoRI19 bp 9 bp 19 bp

23 nt

PCR

RT-PCR

-actin loading control

Figure 1B

0

4000

2000

60001 2 3 4e. i

ii

iii

mRNA

Total RNAloading control

KpnI

gfp shRNA Forward primer

gfp shRNA Reverse primer

Figure 2

100

Cou

nts

FLH-1102101 103 104

0

120

40

80

160

200A BVisible Light UV Light

i

ii

iii

800

1600

2400

0

GFP expre

ssing lin

e

FHVB2-

C17

line

GFP silen

cing l

ine

FHVB2 e

xpre

ssin

g lin

e

Rel

ativ

e in

ten

sity

of

siR

NA

Total RNA loading control

siRNA

Figure 3

(ii)

(iii)

1 2 3 4

(i)

Figure 4

(i)

(ii)

(iii)

13 14 15 16 17 18 Catalase

Aldolase

BA C 1 2 3 M kDa

160

110

73

D 10 11 12 13 14 15 16

17 18 19 20 21 22 23

E

23mer

M 1 2

21mer

0

200 400 600

8001000

120014001600mAU

0.0 5.0 10.0 15.0 20.0 ml

14 1615

0.0 5.0 10.0 15.0 20.0 0

200

400

600

800

1000

1200

1400

1600mAU

A

25.0 ml

Bi

ii

10 11 12 13 14 15 16 17

18 19 20 21 22 23 +ve

C

1514 16

14 15 16 Pool W. Probe

- FHVB2

-DCR-1 PAZ

Figure 5

Figure 6

A

SD (Leu-Trp-His-)SD (Leu- Trp-)

(v) (vi)

YPD

(ii) (iii)

SD (Trp-) SD (Leu-)

(iv)(i)

Scheme

pGADT7+

pGBKT7

pGBKT7

pGA

DT

7 AH109AD (REP)+BD(REP)

AD

(EN

DO

)B

D(F

HV

B

2)

pGBKT7

FHVB2

AD

(PA

Z)

BD

(FH

VB

2)

B (i) (ii)

M 1 2 3 1 2 3 4 M 5 6 116 66 45 35 25

18

(iii)M 1 2 3

INPUT ELUTES FLOW THROUGH FT IEL

Figure 7

M 1 2 3 4 5 6 7

INPUTB GST-FHVB2

MBP-DCR-2-PAZ MBP

EL ELFT FT

11881

52

36

kDa

AAH109

pGAD + pGBD

pGADAD(DCR2PAZ) +BD (FHVB2)

pGBD(FHVB2)

AD(Rep)+ BD(Rep)

pGBD

AD(DCR2 PAZ)+ pGBD

(i) Scheme (ii) SD (Leu-Trp-) (iii) SD (Leu-Trp-His-)

Full Length FHVB2 (107aa)

FHVB2 – C17 (90aa)

N

N

C

C

Figure 8

A

B

C

Sf21 cell line GFP expressing GFP silenced

FHVB2- GFP reverted FHVB2- C17 transfected

00.20.40.60.81.01.2

GFP

GFP silen

ced

GFP reve

rted

FHVB2-

C17

Rel

ativ

e le

vel o

f gf

p m

RN

A

%

%%%

%

( )

( )

( ) ( )

( )

Figure 9

1 2 M 3 4 5 M 6 7

B

INPUT ELUTE F T

(i)

(i) Scheme (ii) SD (Leu-Trp-His-)

A

(iv) SD (Leu-Trp-His-)(iii) Scheme

AD+BD

BD

ADBD(FHV-B2)

AH109

AD(REP)+BD(REP) AD-

DCR1(PAZ)+BD(FHVB2-C17)

AD(PAZ) DCR1+BD(FHVB2)

AD+BD

BD

ADBD(FHV-B2)

AH109

AD(REP)+BD(REP) AD-

DCR2(PAZ)+BD(FHVB2-C17)

AD(PAZ) DCR1+BD(FHVB2)

ELUTE FTINPUT

kDa M 1 2 3

(ii)

36

52

81

118

PAZ

2249 amino acidsN-terminal C-terminal

544aa 606aa 825aa

DUF 283

920aa

HELICASE-C

510aa 1096 aa 1218 aa 1706 aa

RNase III

1919 aa

dsRNA bd

22412175aa aa

MBP-PAZ MBP-ENDONUCLEASE

dm Dicer-1

dm Dicer-2 PAZ

1715 amino acidsN-terminal C-terminal

535aa 554aa 659aa

HELICASES

1aa 838aa 945aa 1187aa

RNase III

1642aa

dsRNA bm

1710aa1646aa

MBP-PAZ

dsRNA bd

A.

B.

C.

Supplementary Figure 1

dm Dicer-1-PAZdm Dicer-2-PAZ

dm Dicer-1-PAZdm Dicer-2-PAZ

dm Dicer-1-PAZdm Dicer-2-PAZ