1 SUPPLEMENTAL MATERIALS: Table of Contents: 1. Supplementary figures (1-6) Pages 2- 1 Fig S1: S1A-S1G - 6 Fig S2: S2 Fig S3: S3A-S3B 2-6 Fig S4: S4 7 Fig S5: S5A-S5G 18 9-1 Fig S2: S2A-S2B 110-1 1 2 2. Supplementary tables (1-3) Pa3ges 61 - Table S1: 1 7 20 Table S2: 18 Table S3: 7 -19 3. Supplemental Methods Pages 220 -2 4. Strobe Checklist Pages 21-24 5 7 2 Supplementary figures: 2./,!34, 3 Shroom3 Actin 2.0 * 2.5 * 2.0 2.0 1.5 2./,!35, 1.5 1.5 1.0 1.0 1.0 -mRNA (normalized ) -mRNA (normalized ) -mRNA (normalized ) 0.5 0.5 0.5 0.0 0.0 Shroom3 0.0 Shroom3 Shroom3 CAGS-TG CAGS-NTG PAX8-RTTA-TG Podocin RTTA-TG PAX8-RTTA-NTG Podocin RTTA-NTG 2./,!3C, $$$$)01$$$$$$$$$$$$$2$$$$$$$$$$$$$$$2$$$$$$$$$!$3"456789$"$ 2./,!36, 4 Synpo Nphs1 Nphs2 +',%(-..'$$$$$$$$$$/.,$$$$$$$$$$.,$$$$$$$$$$.,$$$$$$$$$$$$.,$$$$$$$$$ 3 * 2 Shroom3 1 Gapdh 0 Fold change mRNA (normalized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ig S1E 4 Figure S1E: A disector pair made up of a look-up section and a sample section used for counting podocyte number. Podocyte nuclei profiles are marked with a red asterisk in both the look-up section and the sample section. A red arrow points to the podocyte nuclei profile present in the sample section and not present in the look-up section and is thus counted a Q-. Sections are 1-µm thick stained with toluidine blue. 2./,!32, 5 <A7, A7, !"#$%&!*$C47!?A7,)'@,<A7,&.B%,FE,H%%K0,8$@G,H%*%,-%@,6=>,-8*,J,H%%K0P,;%#*%0%'()(.U%,_`>,.&)/%0,F'M_,%)BO,/*8"#G, 8-,9%*,[email protected],)B.@,!BO.--,0().',)*%,0O8H'P,<8,/$8&%*"$80B$%*80.0V,("R"$)*,)(*8#O+,8*,.'(%*0(.(.)$,&)(*.I,%I#)'0.8',H)0, .@%'(.-.%@,R+,$./O(,&.B*80B8#+,)(,J,H%%K0, Fig S1G 6 Fig S1G: Electron microscope image with superimposed unbiased 2-dimensional counting grid with 10 green parallel counting lines use to quantify length density of the slid diaphragm over peripheral glomerular basement membrane. Section stained with uranyl acetate and lead citrate. Fig S2 7 Fig S2: Glomerulus with superimposed counting grid used to quantify glomerular volume. Sections are 1-µm thick stained with toluidine blue. Fig S3A 8 Cononical Pathway Analysis 40 (Top_Up) Cdh3, Vwf, Snai1, Cldn8, Esrrb, Zhx2, Tgfbr1, Kcnmb1, Synpo2, Cldn7 wiki_XPodNet_−_protein−protein_interactions_in_the_podocyte_expanded_by_STRING 61 (Top_Dn) Wif1, Epha6, Gabra4, Cdc42ep5, Itgb3, Tuba1a, E2f1, Nck2, Adcy1, Slc34a1 24 (Top_Up) Lpar2, Angpt4, Kit, Vwf, Pdgfa, Ppargc1a, Lpar1, Col4a6, Col3a1, Insr wiki_Focal_Adhesion−PI3K−Akt−mTOR−signaling_pathway 26 (Top_Dn) Col1a2, Itgb3, Itgal, Akt1s1, Itgae, Vegfa, Gng2, Col4a1, Col4a2, Il2rg 4 (Top_Up) Col10a1, Col4a6, Col3a1, Cdkn1b PID_AVB3_INTEGRIN_PATHWAY 13 (Top_Dn) Col15a1, Col1a2, Itgb3, Edil3, Vegfa, Col8a1, Col4a1, Csf1, Ptpn11, Tgfbr2 11 (Top_Up) Vwf, Pdgfa, Col4a6, Col3a1, Pgf, Lamb3, Tnn, Actn1, Itgb7, Gsk3b wiki_Focal_Adhesion 19 (Top_Dn) Col1a2, Itgb3, Itgal, Fyn, Itgae, Vegfa, Col4a1, Col4a2, Pdgfb, Itgb5 3 (Top_Up) Sh2d2a, Ptprj, Map2k2 PID_VEGFR1_2_PATHWAY 11 (Top_Dn) Itgb3, Nck2, Fyn, Vegfa, Mapk11, Map2k6, Fes, Ptpn11, Kdr, Mapk14 1 (Top_Up) Cdkn1b wiki_Primary_Focal_Segmental_Glomerulosclerosis_FSGS 13 (Top_Dn) Itgb3, Fyn, Vim, Lmx1b, Plce1, Ptpro, Synpo, Myo1e, Itga3, Tlr4 4 (Top_Up) Capn6, Itgb7, Capn11, Map2k2 change wiki_Integrin−mediated_Cell_Adhesion 13 (Top_Dn) Itgb3, Itgal, Fyn, Itgae, Itgb5, Pak1, Map2k6, Itga3, Pak3, Cav2 Dn 3 (Top_Up) Col10a1, Col4a6, Col3a1 NABA_COLLAGENS 7 (Top_Dn) Col15a1, Col1a2, Col8a1, Col4a1, Col4a2, Col27a1, Col16a1 Up 3 (Top_Up) Ppp2r1b, Atf1, Map2k2 REACTOME_MAP_KINASE_ACTIVATION_IN_TLR_CASCADE 7 (Top_Dn) Cdk1, Mapk11, Map2k6, Nod1, Irak2, Mapk14, Mapk3 5 (Top_Up) Myo3b, Plekhg6, Arhgap32, Ttn, Enah Panther_P00016:Cytoskeletal regulation by Rho GTPase 13 (Top_Dn) Pak1, Stmn1, Limk1, Myh3, Tubb2b, Tubb2a, Pak3, Myh7, Arhgef12, Arhgap1 0 (Top_Up) REACTOME_ACTIVATED_TAK1_MEDIATES_P38_MAPK_ACTIVATION 5 (Top_Dn) Mapk11, Map2k6, Nod1, Irak2, Mapk14 4 (Top_Up) Dkk3, Axin2, Sfrp1, Gsk3b ST_WNT_BETA_CATENIN_PATHWAY 3 (Top_Dn) Wif1, Ankrd6, Dkk2 6 (Top_Up) Pdgfa, Apc2, Actn1, Fgf9, Enah, Map2k2 wiki_Regulation_of_Actin_Cytoskeleton 11 (Top_Dn) Gna12, Pdgfb, Pak1, F2r, Limk1, Pak3, Rras2, Abi2, Nras, Cdc42 6 (Top_Up) Pde1b, Prkar1b, Prkcq, Adcy9, Akap7, Akap11 wiki_G_Protein_Signaling_Pathways 6 (Top_Dn) Pde4b, Adcy1, Gna12, Gng10, Nras, Pde8b 0 3 6 9 −log10(P) 9 Fig S3B Fig S3 A-B: Glomerular and non-Glomerular RNA from DOX-fed Transgenic CAGS-RTTA mice (TG) and Non transgenic littermates (NTG) were extracted using DYNA-bead perfusion and magnetic separation (6-weeks DOX; n=4 in each comparison). RNA-seq was performed on Poly-A selected Ribosomal RNA depleted, total RNA in Illumina NEXTSEQ sequencer (single end, 75 BP reads). Among significantly downregulated genelist (n= 1102 genes; LIMMA test P<0.05), genes uniquely downregulated in the glomerular fraction were identified and ranked by P-value (n=704 genes). (2A) Figure shows significant relevant Meta-pathway analysis terms represented by Up- or down-regulated genes (among top 50 pathways ranked by P-value(see Table S3). We utilized published data21 to identify podocyte specific genes from the glomerular transcriptome. (2B) Figure shows the normalized heat map of top 50 differentially expressed genes. (G4, G5 & CB6ST =TG group; G1, G2, G3, CB6R1G = NTG group) Fig S4 10 Src Kinase SH3 domain P Value Kinase Enrichment Analysis Family Y Y 0.001 N Y 0.001 N Y 0.001 Y Y 0.02 N N 0.02 N N 0.02 N Y 0.001 Y Y 0.03 N N 0.04 Y Y 0.04 Fig S4: Lysates of 293 cells overexpressing SHROOM3 or Control Vector were immuno-precipitated with Anti- V5 (overexpressed and control lysates), Anti-SHROOM3 (control lysates), and separated by PAGE. Lanes were separately analyzed by LCMS. Resulting peptide lists were filtered (see methods). Proteins with > 5 Spectral counts in both Over- expression and Endogenous lanes were ranked by fold change of spectral counts to controls (n=287). This protein list was input into ENRICHR database. Kinase enrichment results are displayed in order of combined score.26 11 !"#$%O$'(!1,6.--%*%'(.)(%@,O"&)', !B*)&R$%,)'@,!.?_,#8@8B+(%,B%$$,$.'%0,!B*)&R$%,)'@,!.?_,#8@8B+(% H%*%,/*8H',.',B8$$)/%'?B8)(%@,JB&, @.0O%0,)(,Sd,nCP,F4G,2!C:!!C,#*8-.$%, )'@,F5G,2!C,\2c,8-,!B*)&R$%,)'@,!.?_, #8@8B+(%0#8@8B+(%0,R+,-$8H,B+(8&%(*+,.0, 0O8H'P,!.?_,0O8H'P,!.?_,#8@8B+(%0,B$"0(%*,&8*%, B8&#)B($+,.'(8,)',)*R.(*)*+,/)(%,)0, .'@.B)(%@P,,FCG,7)(.'/,0(*)(%/+,"0%@, (8,@%-.'%,*)(%0,8-,#8@8B+(%, )#8#(80.0,F6G,)'@,'%B*80.0,F[G,"0.'/, 4'%I.'4'%I.',k,)'@,),U.)R.$.(+,@+%V, *%0#%B(.U%$+P,*%0#%B(.U%$+P,4''%I.',k,(O*%0O8$@, H)0,@%(%*&.'%@,8',95!,B8'(*8$0P,F2G, 98@8B+(%,)#8#(80.0,.'@"B(.8',H.(O, _eo,H:U,j_=_,.'B"R)(.8',-8*,_`&.', )(,NnC,H)0,"0%@,)0,#80.(.U%,B8'(*8$, -8*,4#8#(80.0,)'@,-8*,4#8#(80.0,)'@,4''%I.',k,0().'P, F'MS,.'@%#%'@%'(,%I#%*.&%'(0G,, 12 Fig S5G 0.10 0.08 0.06 0.04 0.02 0.00 Crystal violet absorptiometry (590nm) absorptiometry violet Crystal Si-2 Scramble Fig S5G: Human Scramble and Si-2 podocyte cell lines were grown in collagen-coated 6-well plates (n=3 independent experiments). After complete differentiation at 37-degrees, cells were collagenized and replated in 96 well plates for 24 hours. After washing unattached cells, Crystal violet staining followed by detergent lysis (10%SDS) was performed. Colorimetry was done at 590 nm. Dot blots represent mean colorimetry readings from each experiment. [error-bars=mean±SEM] 13 2./,!J4, C47!,,,,,,,,,,,,,,,<A7,,,,,,,,,,,,<A7 A7,,,,,,,,,,,A7,, !O*88&S, 9O80#O8?<#O03,Fb33dJG, <#O03,F(8()$G, 4B(.', !"#$%P'*$7$8&%*"$)*,#*8(%.',f+0)(%0,8-,)@@.(.8')$,C47!?<A7,D,A7,&.B%,F'M_,%)BOG,H%*%,*"',8',Eo,947[,/%$0, F9O80#O)()0%:9*8(%)0%,.'O.R.(8*0,)@@%@,(8,$+0.0,R"--%*GP,;%0"$(0,8-,.&&"'8R$8((.'/,H.(O,!j;==\SV,9O80#O8,<#O03, Fb33iS:33dJGV,A8()$,<#O03V,D,4B(.',)*%,0O8H'P,AH8,c08-8*&,R)'@0,8-,!O*88&S,)*%,U.0")$.X%@P,AO%,#O80#O8, <9j!31<#O03,@)(),0O8H',O%*%,H%*%,"0%@,-8*,@%'0.(8&%(*+,0O8H',.',2./,JCP,,, 14 Fig S6B Fig S6C 1500 1200 Control-NTG 900 Podocin RTTA-TG 600 NTG 400 350 ACR mcg/mg ACR 300 250 200 150 100 50 0 ADR Week 2 Week4 Week 5 Week 6 Week 8 Baseline 4 weeks 6 weeks Week 1.5 TG Fig S6B-C: DOX-fed Podocin-RTTA mice and littermates (n=5 each group) were injected with Adriamycin (high dose protocol: 18 mg/kg; Arrow in figure). Albuminuria was measured weekly (mcg/mg creatinine). (5B) Figure shows trend of Albuminuria (Dot/Whiskers=Mean/SEM), while (5C) shows representative 20X-images of PAS stained sections. 4/%@,C47!?<A7, 4/%@,C47!?A7, 4/%@,C47!?A7, 2./,!J6 15 , 94!?_`>, c'$)+, 2./,!J[, 7$8&%*"$)*1,,,?,,,,,,,,,,?,,,,,,,,,,?,,,,,,,,,,,?,,,,,,,,,,,,,,,?,,,,,,,,,,?,,,,,,,,,,,?,,,,,,,,,,?,,,,,,,,,,,,,,,q,,,,,,,,,,q,,,,,,,,,,q,,,,,,,,q, 2./,!J2, DOX: 8-wks Aged mice 4/%@1,,,?,,,,,,,,,,?,,,,,,,,,,?,,,,,,,,,,,?,,,,,,,,,,,,,,,q,,,,,,,,,q,,,,,,,,,,q,,,,,,,,,q,,,,,,,,,,,,,,,q,,,,,,,,,,q,,,,,,,,,,q,,,,,,,,q,,,,,,,,,,,,,,,,,,,,,2.0 C47!1,<A7,,,<A7,,,,,A7,,,,,,A7, <A7,,,,,,<A7,,,,,A7,,,,,,A7, ,,,,,,,<A7,,,,,<A7,,,,A7,,,,,,A7, 1.5 1.0 !O*88&S, 0.5 (normalized to NTG) 4B(.', 0.0 DensitometryTubular Shroom3:Actin