1

Lee et al., (2009) Supplemental Methods, Figures, Tables and References.

Supplemental Methods.

Cell lysis buffers: RIPA: 1% TritonX-100, 0.5% Na-deoxycholate, 0.1% SDS, 2 mM EDTA, 150 mM

NaCl, 50 mM Tris-HCl, pH 7.5, 50 mM NaF, 1 mM Na3VO4,1x protease inhibitor coctail [Complete

mini, Roche], 1 mM PMSF, 10 μg/ml leupeptin, 5 μg/ml pepstatin A, 1.7 μg/ml aprotinin, 25 μg/ml

ALLN) supplemented with 1 mM 1,10 phenanthroline and 10 mM N-ethylmaleimide. Triton X-100: 1%

TritonX-100, 1 mM EDTA, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 50 mM NaF, 1 mM Na3VO4,1x

protease inhibitor coctail [Complete mini, Roche], 0.5 mM PMSF, 10 μg/ml leupeptin, 5 μg/ml pepstatin

A, 1.7 μg/ml aprotinin, 25 μg/ml ALLN, 1 mM 1,10 phenanthroline and 10 mM N-ethylmaleimide.

CHAPSO: 50 mM HEPES-KOH, pH 7.4, 100 mM NaCl, 1.5 mM MgCl2 and 0.5% CHAPSO, 5μg/mL

each chymostatin, pepstatin A, leupeptin and antipain. Cell Disruption Buffer: 10 mM HEPES-KOH, pH

7.4, 10 mM KCl, 1.5 mM MgCl2 , 5 mM EDTA, 5 mM EGTA, 250 mM sucrose, 5 mM DTT, 1x protease

inhibitor coctail [Complete mini, Roche], 1 mM PMSF, 10 μg/ml leupeptin, 5 μg/ml pepstatin A, 1.7

μg/ml aprotinin, 25 μg/ml ALLN, 1 mM 1,10 phenanthroline and 10 mM N-ethylmaleimide. Membrane

Solubilization Buffer: 10 mM Tris-HCl, pH 7.3, 100 mM NaCl, 1% SDS, 1 mM EDTA and 1 mM

EGTA. Nuclear Extraction Buffer: 20 mM HEPES-KOH, pH 7.6, 2.5% glycerol, 420 mM NaCl, 1.5 mM

MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1x protease inhibitor coctail, 1 mM PMSF, 10 μg/ml

leupeptin, 5 μg/ml pepstatin A, 1.7 μg/ml aprotinin, 25 μg/ml ALLN, 1 mM 1,10 phenanthroline and 10

mM N-ethylmaleimide.

Primer sequences used for real-time PCR (5’ to 3’): HMGCR for CAG GGA ACC TCG GCC TAA

TG, rev CGG CGA ATA GAT ACA CCA CGC; INSIG-1 for ATC GTT CCA GAA GTG GCC TTG,

rev CTA TGT TCC CCA CCG TGA CG; INSIG-2 for ATG TGA CGC TCT TTC CAC CTG, rev CAC

CGC ATT ACA CTG GAC CAC; MVLK for TGG ACC AAG GAG GAT TTG GAG, rev TGC GAG

GGA CTT TGG TGT TG; MAC30 for TTT GCG AGC TTG TGT TTC AGC, rev TGT CCC TTG AAA

CCA CTG GC; LDLR for GAC CGG AGC GAG TAC ACC AG, rev TGG GTG CTG CAG ATC ATT

2

CTC; SCD1 for CAC TTG GGA GCC CTG TAT GG, rev AGC CGA GCT TTG TAA GAG CG;

SREBF1 for GCG CAA GGC CAT CGA CTA C, rev CAG TCT TCA CGC CCT CCA TG; SREBF2 for

TTC AGC ACC ACT CCG CAG AC, rev CAT TGG CCG TTT GTG TCA GC; FASN for CAC AGT

CAC CAT CTC GGG ACC, rev CTC CCG GAT CAC CTT CTT GAG; DHCR7 for TCT CGC CCA

CCA TCA TCT, rev CGC CCA TTG AAG AAC AGC; SQLE for CGT GCT TGG CTC TGC TTT, rev

CCA TTT ACA ACC TGG GCA TC; HMGCS for GCT GCT GTC TTC AAT GCT GT, rev ACT GCT

CCA ACT CCA CCT GT.

Affymetrix microarray analysis of gene expression. Duplicate cultures of HEK293FI cells

carrying TRC8 or the RING mutation C547S;C550S were induced with doxycycline for 20 h.

RNA was purified using the RNeasy kit (QIAGEN, Inc., Valencia, CA). All labeling and

hybridizations were carried out in the UCHSC cDNA Microarray Core using standard protocols.

Data were analyzed using the GeneChip Operating Software (GCOS) v1.4 (Affymetrix, Santa

Clara, CA). CEL files were imported to GeneSpring v7.2 (Agilent, Redwood City, CA) and

analyzed for probe level using the GC-Robust Multi-array Average (GC-RMA) algorithm.

Signals were normalized to TRC8wt expressing cells and compared to TRC8CAIC to identify

changes. Filters removed genes with low raw signals (<10), poor signal consistency (1.5-fold

outside a regression curve) and genes whose expression did not change. We tested a final 1,921

genes using a p-value cutoff of 0.15 for the Benjamini and Hochberg False Discovery Rate

multiple testing correction (1). 876 genes were ultimately found to have statistically significant

differences between the two conditions.

Microarray Data Management in GeneSpring. Initial data quality assessment was performed

using GeneChip Operating Software (GCOS) v1.4 (Affymetrix, Santa Clara, CA). Resulting

CEL files were imported to GeneSpring v7.2 (Agilent, Redwood City, CA) for analysis using

their native probe level GC-Robust Multi-array Average (GC-RMA) algorithm (2-4). GC-RMA

3

consists of three steps: an initial background noise/non-specific binding correction is performed

using the G-C content to estimate hybridization affinities of the individual targets that comprise a

set of probes for any given gene. In the first step, the affinity corrected mis-match (MM)

hybridization scores are subtracted from perfect match (PM) hybridization scores for every target

of a given probe set. Secondly, a quantile non-linear normalization is performed that corrects for

bias among arrays in the full data set, such that each array can be directly compared to any other.

Finally the quantile normalized probe level data, derived from the initial background noise/non-

specific binding correction step, are summarized as a single expression measurement for that

given probe set.

A text file was generated using the GeneSpring GC-RMA algorithm that represents probe

level summary data from each array in the sample set. This ‘master’ data file was then arranged

by parameter such that data from individual chips were considered as duplicates. For example,

the two wild-type samples were considered as ‘WT’ duplicates and likewise for the two CAIC

mutation samples following the GC-RMA. Raw values below 1.0 were set to 1.0 and each

measurement was then divided by the 40th percentile of all measurements in that sample. The

percentile was calculated with all raw measurements above 1.0. Specific samples were

normalized against the mean of the wild-type duplicate samples. In other words, each

measurement for each gene in all samples was divided by the mean of that gene's measurements

in the corresponding control samples. Finally, the log (base 2) normalized intensity values were

plotted in GeneSpring in order to view relative changes in gene expression between the two

conditions.

In order to ascertain reliable expression levels, filters were used to remove genes with

expression data that did not meet specific criteria. Any gene that scored below 10 raw data units

4

in both conditions (wild-type or CAIC mutant) were removed leaving 30,342 of the initial 54,675

genes on the Affymetrix Hu133_2.0 array. Using this remainder list of genes, the individuals of

each duplicate data set were plotted against each other to fit a linear regression. Using the range

of gene expression in those duplicates, genes with a range that exceeded 1.50 fold outside the

linear regression curve for either the wild-type or the CAIC mutant duplicate sets were removed

(5,545 or 1,783 respectively) resulting in a list of 23,521 genes.

The next filter removed genes that were equal between the wild-type and CAIC duplicate

conditions by at least 20% of the mean (20,962 genes). The resultant list of 2,559 genes has a

reasonable degree of confidence for expression changes between the two conditions and was

sorted for fold differences using a 1.33 fold cutoff value yielding 970 genes. The possibility

existed that other changing genes were excluded during the initial correlation test due to very

low expression values that may not have been precisely linear within the whole data set;

therefore the lists excluded from the correlation test were also examined for genes differential by

at least 20% of their means and by 1.33 fold change, thereby recovering an additional 951 genes.

After combining the highly expressed 970 genes with the low expressing 951 genes, we

performed parametric statistical testing. Variances were calculated using a cross-gene error

model that grouped the samples according to previously furnished biologic information (i.e.

which construct each sample contained). The cross-gene error model was active. A p-value

cutoff 0.15 was used for Benjamini and Hochberg False Discovery Rate multiple testing

correction (1). This restriction tested 1,921 genes and about 15.0% of the identified genes would

be expected to pass the restriction simply by chance. We found 876 genes from this combined

changers list (1,921) which had statistically significant differences when grouped by the

parameter 'Construct'.

5

Supplemental Figures.

Supplemental Figure S1. Multiple sequence alignment of SSDs from SCAP, HMGCR and

TRC8. Sequence alignments were established using Clustal W followed by manual adjustments

(5). Identities are shaded with black, similar amino acids are shaded in grey. Boxes indicate the 5

transmembrane segments (TM) comprising the SSDs of SCAP and HMGCR (top) and TRC8

(bottom). A conserved tyrosine residue at position 32 of TRC8, implicated in SSD function of

SCAP and HMGCR, is indicated by the arrow.

Supplemental Figure S2. TRC8 is stabilized by sterol deprivation in CHO cells. CHO cells

were transiently transfected with pcDNA5/FRT/TO-TRC8-HA expression construct and then

cultured in sterol-depletion media over-night to accumulate TRC8. At time 0, media were

replaced with sterol-depleted or replete media, as indicated, cycloheximide (CHX) was added

and the indicated time points harvested for Western analysis. Addition of cholesterol / 25-HC

resulted in destabilization of exogenous TRC8 by 5-8h (lanes 4-5).

6

Figure S3. TRC8 knockdown using siRNA or shRNA. (A) CHO-FI cells expressing TRC8-

HA were transfected with the indicated siRNAs (Ambion, Inc.) and analyzed 72h later by

Western blot. Controls included scrambled (scram) and siRNA targeting GAPDH. GAPDH

knock-down was verified in control cells with anti-GAPDH antibody while actin verified equal

loading. (B) HEK293FI-vector controls were transfected with the indicated siRNAs and analyzed

for endogenous TRC8 and GAPDH mRNA by qRT-PCR 72h later. Error bars; +/- std. dev.

(n=3).(C) Amb-27 shRNA sequences targeting TRC8 were cloned into pSuperior.puro and stably

transfected into HEK293FI cells to generate an inducible knockdown line, TRC8 k/d. Vehicle or

dox-treated TRC8 k/d cells (in triplicate) were analyzed for TRC8 mRNA at the indicated times

following dox addition by realtime RT-PCR. Normalization was against β-actin; differences

between 0 and 96h samples were highly significant (**p = 0.007, Welch’s t-test).

7

Figure S4. Reciprocal coimmunoprecipitation of INSIG and TRC8 and sterol independence

of interaction. (A) HEK293 cells containing vector, dox-inducible TRC8-HA or ΔRING mutant

were transfected with pCMV-INSIG-1-myc (1), pCMV-INSIG-1-myc (K156R, K158R double

mutant; kk) or pCMV-INSIG-2-myc (2) and 24h later treated with dox (100ng/ml) overnight to

induce TRC8. MG132 was added for the final 4h and lysates prepared in RIPA buffer. Pre-

cleared lysates were immunoprecipitated with anti-myc 9E10 beads to precipitate INSIG and the

pellets analyzed for TRC8-HA. IgG heavy chain is the common band on the IP filter. Lysates

were tested for the indicated proteins and a Coomassie-stained gel verified equal inputs. (B)

CHO-FI cells carrying vector (vec), TRC8-HA, or ΔRING mutant were transfected with INSIG-1.

Twenty four hours post transfection, cells were switched to sterol-replete (+) or sterol-deprivation (-)

media, as indicated. After 20h further incubation, lysates were prepared in CHAPSO buffer and subjected

to anti-HA immunoprecipitation. IP pellets were analyzed by Western blot for INSIG (myc) and TRC8

(HA). Input samples verified transfected INSIG-1, TRC8-HA expression and loading (actin) while

HMGCR verified efficacy of sterol-depletion.

8

Figure S5. Time course of SREBP loss induced by TRC8. HEK293FI cells carrying vector or

TRC8-HA were dox-induced (100ng/ml) over a 48 hour time-course, as indicated, and analyzed

for pre-SREBP-1 and -2 and TRC8-HA. Asterisk identifies a constant background band.

Figure S6. SREBP precursors are processed in response to sterol-deprivation in HEK293

cells. Media were replaced in duplicate cultures of 293FI vector cells with normal media

containing 8% FCS (C, lanes 1-2) or with sterol-depleted media (see Methods). After 12h sterol-

deprivation, either cholesterol (+; 12.5 μg/ml / 10 μg/ml 25-HC), or mevastatin/mevalonate (-;

50 μM each) were added and incubation continued for 20 h. Cells were harvested, lysed and

fractionated into membrane and nuclear compartments (6) (see Methods). Membrane and nuclear

fractions were analyzed by Western for SREBP-1 (2A4 antibody), SREBP-2 (1D2 antibody)

9

along with CREB-1 (nuclear control) and calnexin (membrane control). Excess sterols caused

accumulation of SREBP precursors in the membrane fractions (lanes 3-4), while sterol-depletion

had the opposite effect (lanes 5-6). The processed N-terminus of SREBP-1/2 was increased in

the nuclear fractions by sterol-deprivation, unambiguously identifying these bands as nucSREBP

(lanes 5-6).

Figure S7. TRC8-induced SREBP loss does not increase nucSREBPs. Duplicate cultures of

293FI cells carrying vector, TRC8-HA or TRC8-ΔRING were grown in normal FCS medium

with or without dox induction (100 ng/ml), as indicated. After 24h, cells were processed into

membrane and nuclear fractions, and analyzed for precursor and nuclear forms of SREBP-1/2

using 2A4 (SREBP-1) and 1D2 (SREBP-2) antibodies to detect both precursor and nuclear

forms. Only wild type TRC8 affected precursor SREBP levels and nuclear levels were not

increased.

10

Table S1. Lipid homeostasis genes repressed by TRC8 expression

Fold GENE Known SREBP Affymetrix Accession

Change SYMBOL DESCRIPTION Targets1 ID Number

-3.13 SCD1 stearoyl-CoA desaturase (delta-9-desaturase) YES 211708_s_at NM_005063

-2.94 INSIG1 Insulin induced gene 1 YES 201625_s_at NM_005542

-2.87 LDLR low density lipoprotein receptor (familial hypercholesterolemia) YES 202068_s_at NM_000527

-2.03 ABCG1 ATP-binding cassette, sub-family G (WHITE), member 1 PROBABLE 204567_s_at NM_004915

-1.93 SC4MOL Sterol-C4-methyl oxidase-like YES 209146_at NM_006745 -1.91 DHCR7 7-dehydrocholesterol reductase YES 201790_s_at NM_001360

-1.90 HMGCS 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) YES 205822_s_at NM_002130

-1.84 SQLE Squalene epoxidase YES 213577_at NM_003129 -1.72 IPP1 isopentenyl-diphosphate delta isomerase 1 YES 208881_x_at NM_004508 -1.72 OSBPL8 Oxysterol binding protein-like 8 228986_at NM_020841

-1.71 HMGCR 3-hydroxy-3-methylglutaryl-Coenzyme A reductase YES 202540_s_at NM_000859

-1.69 SCD2 stearoyl-CoA desaturase 5 YES 224018_s_at NM_024906

-1.59 FDFT1 farnesyl-diphosphate farnesyltransferase 1= squalene synthase YES 210950_s_at NM_004462

-1.57 STARD4 START domain containing 4, sterol regulated YES 226390_at NM_139164

-1.56 ACSL4 Acyl-CoA synthetase long-chain family member 4 YES 1557418_at NM_004458

-1.55 FADS2 fatty acid desaturase 2, same as delta 6 FADS YES 202218_s_at NM_004265

-1.51 SREBF2 Sterol regulatory element binding transcription factor 2 PROBABLE 201247_at NM_004599

-1.50 DHCR24 24-dehydrocholesterol reductase PROBABLE 200862_at NM_014762 -1.47 AMACR Alpha-methylacyl-CoA racemase 209425_at NM_014324 -1.45 MVLK mevalonate kinase (mevalonic aciduria) YES 215649_s_at NM_000431

-1.40 SC5DL Sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, fungal)-like YES 215064_at NM_006918

1. Based upon Horton et al., (2003)

Supplemental Table S1. The complete list of lipid metabolic and regulatory genes affected in a

statistically significant manner by 20h expression of wild type TRC8 compared to C547S;C550S

RING-mutant. The lipid-related genes all showed reduced expression and represented about 4%

of nearly 500 suppressed genes.

11

Figure S8. Enhanced expression of SREBP target genes upon sterol-deprivation in TRC8

knocked-down cells. RNA was isolated from HEK293/TRC8 knockdown cells containing a

dox-induced shRNA against TRC8. Duplicate cultures were treated for 4 days with vehicle or

dox (+) to k/d TRC8. Media were changed 20h prior to harvest either to fresh 5% FCS or fresh

sterol-depletion media (5% LPDS, 10 μM mevastatin / 50 μM mevalonate) to activate the

SREBP pathway. RNAs were analyzed by qRT-PCR for expression levels of SREBP target

genes including MVLK, SCD1, INSIG-1 and LDLR. Controls (open bars); dox-induced

knockdown (grey bars); sterol-deprived (striped bars); TRC8 knockdown and sterol-deprived

(black bars). Expression is reported relative to GAPDH and normalized to control cultures (open

bars) for each gene; Error bars indicate SEM (n = 4); *p<0.05, Student’s t-test; NS, not

significant. All four genes show higher expression trends in the knockdown samples that have

been sterol-deprived, although two do not reach significance.

12

Figure S9. Time-dependence of enhanced SREBP target gene expression. Further examples

of SREBP target genes whose expression is altered upon TRC8 knockdown in sterol-depleted

cells. Samples analyzed are the same as shown in Fig 4E. The trend of increased expression with

TRC8 knockdown after 12 or 24h of sterol-deprivation is clear, although several time points do

not achieve statistical significance (NS); **p<0.01by Student’s t-test.

Figure S10. Mitotic recombinant clones expressing DTrc8 in imaginal discs. Larvae were

generated which carried the AYG cassette along with heat-shock-inducible FLP recombinase,

UasGFP and UasDTrc87.1. Wing imaginal discs were dissected from third instar larvae which

had been previously heat shocked at the hatchling stage. The yellow blocking sequence, flanked

13

by FRT homologous recombination sites, was removed by heat-shock-induced FLP recombinase

in a stochastic fashion, creating patches of GAL4-expressing tissue. Imaginal discs containing

GFP+ clones were dissected from late third instar larvae, fixed and incubated with primary and

secondary antibodies as described (7). Primary antibody was a 1:100 dilution of anti-MYC mAb

9E10 (Sigma) detected using a 1:1000 dilution of Cy3 conjugated goat anti-mouse antibodies.

After washing, wing and eye discs were dissected and mounted in PermaFluor. Fluorescence

images were captured using the Zeiss AxioSkop system or a Zeiss confocal microscope. (A) GFP

fluoresence reveals a recombinant clone in a wing disc. (B) anti-MYC staining verifies DTrc8-

myc expression in the same clone. (C) merged image.

Figure. S11. Expression of DTrc8 phenocopies Minute mutations. (A, B) SEM image (500x) of

normal adult cuticle (A) and cuticle containing a DTrc8-expressing clone (B) from dorsal surface of

notum. The clone in (B) is demarcated by black lines, within which small bristles (microchaetes) were

14

shrunken and deformed, while the regular arrays of bristles were disrupted. (C) Scutellar bristles

(macrochaetes) arising within a DTrc8-expressing clone (circled at base) were short compared to bristles

arising from normal cuticle (arrowheads). (D, E) Macrochaetes were abnormally thin when arising from

DTrc8-expressing clones (E) compared to normal cuticle (D).

Supplemental references.

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discovery rate controlling procedures. Bioinformatics (Oxford, England) 2003;19(3):368-75.

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density oligonucleotide array data based on variance and bias. Bioinformatics (Oxford, England)

2003;19(2):185-93.

3. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix

GeneChip probe level data. Nucleic Acids Res 2003;31(4):e15.

4. Irizarry RA, Hobbs B, Collin F, et al. Exploration, normalization, and summaries of high density

oligonucleotide array probe level data. Biostatistics 2003;4(2):249-64.

5. Gemmill RM, West JD, Boldog F, et al. The hereditary renal cell carcinoma 3;8 translocation

fuses FHIT to a patched-related gene, TRC8. Proceedings of the National Academy of Sciences of the

United States of America 1998;95(16):9572-7.

6. Sever N, Yang T, Brown MS, Goldstein JL, DeBose-Boyd RA. Accelerated degradation of HMG

CoA reductase mediated by binding of insig-1 to its sterol-sensing domain. Molecular cell 2003;11(1):25-

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