Supplementary Fig. S1. (A) Predicted secondary structures of mature miR-21 and its target in thePTEN 3’ UTR of human (left), mouse (right) and rat (bottom) are shown. (B) Predicted secondarystructures for the seed sequence of miR-21 and its target in the PTEN 3’ UTR of human (left), mouse(right) and rat (bottom) are shown. miR-21 sequence is shown in green. PTEN 3’ UTR mRNA target isshown in red. Minimum free energy (mfe) for the predicted structures is shown.

Rat, mfe: -14.4 kcal/mol

Mouse, mfe: -13.7 kcal/molHuman, mfe: -15.2 kcal/molA

Human, mfe: -8.8 kcal/mol Mouse, mfe: -9.8 kcal/mol

Rat, mfe: -11.5 kcal/mol

B

LUCIFERASEPTEN 3’ UTR

SV40 PROMOTER

SV40 LATEPOLY A SIGNAL

Supplementary Fig. S2. Structure of the PTEN 3’ UTR-Luc reporter construct. A fragment of thecDNA coding for PTEN 3’ UTR (865 bps) containing miR-21 response element of PTEN is fused to the3’ of the SV40 promoter-driven luciferase coding sequence.

Supplementary Fig. S3. Expression of miR-21 in pCMV-miR-21-transfected rat mesangial cells forthe results presented in Fig. 4. (A) Mesangial cells were transfected with pCMV-miR-21 or vectorexpressing scrambled RNA in parallel to the experiment shown in Fig. 4A. (B) Mesangial cells weretransfected with pCMV-miR-21 or vector expressing scrambled RNA in parallel to the experimentshown in Fig 4B - 4D. Total RNAs from these cells were used to detect mature miR-21 and U6 RNA byreal time qRT-PCR as described in the Materials and Methods of the text.

0

2

4

6

8

miR-21 - +

Scrambled + -

miR

-21/

U6

A

0

1

2

3

4

miR-21 - +

+ -

miR

-21/

U6

Scrambled

B

Supplementary Fig. S4. Structure of the miR-21 Sponge expression vector. Seven copies of anti-miR-21sequence each with one bulge were cloned downstream of green fluorescence protein (GFP) cDNA.Sequence of the anti-miR-21 bulge with complementarity with 3’ UTR of PTEN mRNA is shown at thebottom box. Arrow indicates the RNA PolII-driven transcription from cytomegalovirus (CMV) promoter.

CMV

Pol IIGFP

microRNA binding sites(bulged)

Poly A

3’-AGUUGUAGUCAGAC

UAUUCGAU-5’ miR-21

5’-UCAACAUCAGGAC

AUAAGCUA-3’Sponge

Supplementary Fig. S5. Expression of anti-miR-21 prevents the high glucose-induced inhibition of PTEN3’ UTR-Luc reporter activity. (A) Expression of miR-21 in anti-miR-21-transfected rat mesangial cells forthe results presented in panel B. Total RNA was used to detect mature miR-21 and U6 RNA using realtime qRT-PCR described in the text. (B) Rat mesangial cells were transfected with PTEN 3’UTR-Lucalong with scrambled RNA or anti-miR-21 followed by incubation with 25 mM glucose (HG) or 5 mMglucose plus 20 mM mannitol (NG) for 24 hours. The cell lysates were used for luciferase activity asdescribed in the Materials and Methods of the text. Mean ± SE of 6 measurements is shown. *p < 0.05 vsNG; #p < 0.05 vs NG; **p < 0.05 vs HG.

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U6

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ativ

e Lu

cife

rase

Act

ivity

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itrar

y U

nit)

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NG NGHG HG

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--++

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--++

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GSK3β

Actin

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**

pGSK

3β/G

SK3β

DScr

Anti-miR-21

NG NGHG HG

- - + +

--++

Supplementary Fig. S6. Expression of anti-miR-21 prevents PTEN downregulation and inhibitsdownstream signaling induced by high glucose. (A) Inhibition of mature miR-21 expression in anti-miR-21-transfected mesangial cells for the results described in panels B - D. Total RNA was used to detectmature miR-21 using real time qRT-PCR as described in the Materials and Methods of the text. (B - D)Mesangial cells were transfected with anti-miR-21 or scrambled RNA followed by incubation with highglucose as described in the legend of Supplementary Fig. S5B. The cell lysates were immunoblotted withPTEN, phospho-Akt, Akt, phospho-GSK3β, GSK3β and actin antibodies as indicated. Histograms at thebottom of each panel represent quantification of the protein bands. n = 6. *p < 0.001 vs NG; **p < 0.001vs HG in paenl B. *p < 0.01 vs NG; **p < 0.01 vs HG (n = 6) in panel C. *p < 0.05 vs NG; **p < 0.05 vsHG (n = 6) in panel D.

0.51

1.52

2.53

3.54

0miR-21 - +

Scrambled + -

miR

-21/

U6

Supplementary Fig. S7. Expression of miR-21 in the pCMV-miR-21-transfected mesangial cells for theresults presented in Fig. 6A. Mesangial cells were transfected with pCMV-miR-21 or vector expressingscrambled RNA in parallel to the experiment shown in Fig. 6A. Total RNAs from these cells were used todetect mature miR-21 and U6 RNA by real time qRT-PCR as described in the Materials and Methods ofthe text.

pPRAS40

Actin

PRAS40

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A

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-21/

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NG NGHG HG

- - + +

--++

*

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pPR

AS4

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AS4

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Supplementary Fig. S8. Expression of anti-miR-21 inhibits high glucose-stimulated phosphorylation ofPRAS40. (A) Inhibition of endogenous miR-21 levels in the anti-miR-21-transfected mesangial cells in anexperiment performed in parallel to that described in panel B. Total RNA was used to detect endogenousmiR-21 using real time qRT-PCR as described in the Materials and Methods of the text. (B) Mesangialcells were transfected with anti-miR-21 or scrambled RNA followed by incubation with high glucose asdescribed in the legend of Supplementary Fig. S5B. The cell lysates were immunoblotted with phospho-PRAS40, PRAS40 and actin antibodies as indicated. The histogram at the bottom shows quantification ofthe protein bands. n = 4. *p < 0.001 vs NG; **p < 0.001 vs HG.

012345678

miR-21 - +

Scrambled + -

miR

-21/

U6

Supplementary Fig. S9. Expression of miR-21 for the results presented in Fig. 6C. Mesangial cells weretransfected with pCMV-miR-21 or vector expressing scrambled RNA in parallel to the experiment shownin Fig. 6C. Total RNAs from these cells were used to detect mature miR-21 and U6 RNA by real timeqRT-PCR as described in the Materials and Methods of the text.

A

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U6

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pS6K

S6K

Actin

pS6K

/S6K

*

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Scr

Anti-miR-21

NG NGHG HG

- - + +

--++

B

Supplementary Fig. S10. Expression of anti-miR-21 inhibits high glucose-stimulated phosphorylation ofS6 kinase. (A) Inhibition of endogenous miR-21 levels in the anti-miR-21-transfected mesangial cells inan experiment performed in parallel to that experiment described in panel B. Total RNAs were used todetect endogenous miR-21 using real time qRT-PCR as described in the Materials and Methods of the text.(B) Mesangial cells were transfected with anti-miR-21 or scrambled RNA followed by incubation withhigh glucose as described in the legend of Supplementary Fig. S5B. The cell lysates were immunoblottedwith phospho-S6 kinase, S6 kinase and actin antibodies as indicated. The histogram at the bottom showsquantification of the protein bands. n = 6. *p < 0.001 vs NG; **p < 0.001 vs HG.

0

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B

- + - +miR-21-+-+Vector

LY 294002 ++--

- + - +miR-21-+-+Vector

MK-2206 ++--

miR

-21/

U6

miR

-21/

U6

Supplementary Fig. S11. Expression of anti-miR-21 for the results presented in Fig. 6E and 6F.Mesangial cells were transfected with pCMV-miR-21 or vector expressing scrambled RNA in parallel tothe experiment shown in Fig. 6E and 6F. The cells were incubated with Ly294002 (panel A) or MK-2026(panel B) as described in the Fig. 6E and 6F. Total RNAs from these cells were used to detect mature miR-21 and U6 RNA by real time qRT-PCR as described in the Materials and Methods of the text.

A B

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45

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0123

4567

miR

-21/

U6

miR

-21/

U6

miR-21 - +

Scrambled + -

miR-21 - +

Scrambled + -

Supplementary Fig. 12. Expression of miR-21 in pCMV-miR-21-transfected mesangial cells for theresults described in the Fig. 7A and 7B. Mesangial cells were transfected with pCMV-miR-21 or vectorexpressing scrambled RNA in parallel to the experiment shown in Fig. 7A and 7B, respectively. TotalRNAs from these cells were used to detect mature miR-21 and U6 RNA by real time qRT-PCR asdescribed in the Materials and Methods of the text.

GFP

GAPDH

ANG NGHG HG

miR-21 Sponge - - + +

Vector --++

GFP

GAPDH

BNG NGHG HG

miR-21 Sponge - - + +

Vector --++

Supplementary Fig. 13. Expression of miR-21 Sponge for the results presented in the Figs. 7C and 7D.Mesangial cells were transfected with miR-21-Sponge or vector plasmid followed by incubation with highglucose as described in the legend of Fig. 5C. Total RNAs were used in RT-PCR for the detection of GFPmRNA, which serves as the surrogate for the expression of endogenous miR-21 neutralizing “Sponge”sequence.

35S

Met

hion

ine

Inco

rpor

atio

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ertro

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tein

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l)

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U6

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Supplementary Fig. 14. Expression of anti-miR-21 blocks high glucose-stimulated protein synthesis andhypertrophy in mesangial cells. (A and C) Inhibition of endogenous miR-21 levels in the anti-miR-21-transfected mesangial cells in parallel to the experiment performed in panel B and D, respectively. TotalRNAs were used to detect endogenous miR-21 using real time qRT-PCR as described in the Materials andMethods of the text. (B and D) Mesangial cells were transfected with anti-miR-21 or scrambled RNAfollowed by incubation with high glucose as described in the legend of Supplementary Fig. S5B. Proteinsynthesis was determined as described in the Materials and Methods in the text as 35S-incorporation (panelB). Mean ± SE of 6 measurements is shown. *p < 0.01 vs NG; **p < 0.01 vs HG. Hypertrophy ofmesangial cells were determined as a ratio of total protein content to cell number as described in theMaterials and Methods of the text (panel D). Mean ± SE of 6 measurements is shown. *p < 0.001 vs NG;**p < 0.05 vs HG.

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U6

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miR

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U6

Supplementary Fig. 15. Expression of miR-21 for the results presented in the Figs. 7E - 7H. Mesangialcells were transfected with CMV-miR-21 or vector plasmid followed by incubation with Ly294002(panels A and B for Figs. 7E and 7F, respectively) or MK-2026 (panels C and D for Figs. 7G and 7H,respectively). Total RNAs were used in real time RT-PCR for the detection of miR-21.

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-21/

U6

01234567

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-21/

U6

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Fibr

onec

tin/ a

ctin

Fibronectin

Actin

*

**

C

Scr

Anti-miR-21

NG NGHG HG

- - + +

--++

Supplementary Fig. 16. (A) Expression of miR-21 in pCMV-miR-21-transfected mesangial cells for theresults presented in Fig. 8A. Total RNAs from pCMV-miR-21-transfected mesangial cells were used todetect expression of mature miR-21 and U6 RNAs using real time qRT-PCR as described in the Materialsand Methods of the text. (B) Expression of miR-21 in anti-miR-21-transfected mesangial cells for theresults described in the panel C. (C) Expression of anti-miR-21 inhibits high glucose-stimulatedfibronectin expression. Mesangial cells were transfected with scrambled RNA or anti-miR-21 followed byincubation with high glucose as described in the legend of Fig. S5B. The cell lysates were immunoblottedwith fibronectin and actin antibodies as indicated. The histogram at the bottom shows quantification of theprotein bands. n = 6. *p < 0.001 vs NG; **p < 0.001 vs HG.

A

miR

-21/

U6

01234567

miR-21 - +

Scrambled + -

B

GFP

GAPDH

NG NGHG HG

miR-21 Sponge - - + +

Vector --++

Supplementary Fig. 17. (A) Expression of miR-21and miR-21 Sponge for the results presented in Figs.8C and 8D. Mesangial cells were transfected with pCMV-miR-21 (panel A) or miR-21 Sponge (panel B).Total RNAs were used to detect expression of miR-21 and U6 by real time RT-PCR (panel A) or to detectexpression of GFP mRNA as a surrogate for miR-21 Sponge expression (panel B)

A

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-21/

U6

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ativ

e Lu

cife

rase

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ivity

(Arb

itrar

y U

nit)

*

**

B

Scr

Anti-miR-21

NG NGHG HG

- - + +

--++

Supplementary Fig. 18. Expression of anti-miR-21 inhibits high glucose-stimulated fibronectinexpression. (A) Inhibition of miR-21 in anti-miR-21-transfected mesangial cells for the results described inpanel B. Total RNAs isolated from anti-miR-21-transfected mesangial cells were used to detect maturemiR-21 using real time qRT-PCR as described in the Materials and Methods of the text. (B) Mesangialcells were cotransfected with Fibro-Luc and anti-miR-21 or scrambled RNA followed by incubation withhigh glucose as described in Supplementary Fig. S5B. The cell lysates were used for luciferase activity asdescribed in the Materials and Methods of the text. Mean ± SE of 6 measurements is shown. *p < 0.05 vsNG; **p < 0.05 vs HG.

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-21/

U6

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LY 294002 ++--

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miR

-21/

U6

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MK-2206 ++--Supplementary Fig. 19. Expression of miR-21 for the results presented in the Figs. 8E and 8F. Mesangialcells were transfected with CMV-miR-21 or vector plasmid followed by incubation with Ly294002 (panelA for Fig. 8E) or MK-2026 (panel B for Fig. 8F). Total RNAs were used in real time RT-PCR for thedetection of miR-21.

Vector

miR-21 Sponge

NG NGHG HG

- - + +

--++

GFP

A

Vector

miR-21 Sponge

NG NGHG HG

- - + +

--++

GAPDH

GFP

GAPDH

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Vector

miR-21 Sponge

NG NGHG HG

- - + +

--++

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GAPDH

C

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miR-21 Sponge

NG NGHG HG

- - + +

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GFP

GAPDH

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miR-21 Sponge

NG NGHG HG

- - + +

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GAPDH

E

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NG NGHG HG

- - + +

--++

GFP

GAPDH

F

Vector

miR-21 Sponge

NG NGHG HG

- - + +

--++

GFP

GAPDH

G

Supplementary Fig. 20. Expression of miR-21 Sponge in PTE cells for the results described in the Figs.9C - 9L. Total RNAs prepared from the parallel experiments described in these panels were used to detectthe expression of GFP and GAPDH mRNAs using RT-PCR. Expression of GFP is used as a surrogate forthe expression of miR-21 Sponge. Panel B represents for the Figs D - F. Panel C represents for the Figs.8G and 8H. Panels D - G represent Figs. 8I - 8L, respectively.

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Supplementary Fig. 21. Expression of miR-21 for the results presented in the Figs. 10A - 10H. PTE cellswere transfected with CMV-miR-21 or vector plasmid followed by incubation with Ly294002 (for Figs.8A, 8C, 8D and 8G) or MK-2026 (for Figs. 8B, 8E, 8F and 8H). Total RNAs were used in real time RT-PCR for the detection of miR-21.