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Supplementary Table 1. MiRs significantly deregulated in bortezomib-resistant cells*
MiRNA
92-1
29c
29b
301a
19a
21
30e
192
660
27a
Fold-decrease
11.3
7.2
6.7
4.9
3.9
2.3
2.3
2.1
2.0
1.8
MiRNA
92b
3185
1908
1469
2861
1915
939
762
638
149*
1290
1202
3196
129
1207
1246
Fold-increase
27.9
19.8
17.0
14.6
13.6
13.3
13.0
13.0
12.4
11.4
9.5
9.2
8.0
5.7
3.5
3.5
*p-values < 0.05; fold change threshold of > 1.5
Supplementary Table 1. RNA was isolated from RPMI8226 cells using the RNeasy mini kit
(Qiagen, Valencia, CA), quality checked using an Agilent 2100 Bioanalyzer, labled with
flashtag™ biotin HSR (Affymetrix, Santa Clara, CA) and hybridized to to GeneChip® miR 2.0
arrays (Affymetrix) that included probe sets for 15,644 total mature miRs, 2,202 human premiRs and 2,334 snoRNAs and scaRNAs. Samples were added to hybridization chambers that
contained biotin-labeled LMW RNAs and 20X hybridization controls (1.5pM bioB, 5pM bioC,
25pM bioD, 100pM cre), 10% DMSO, 27.5% formamide, and 1X hybridization buffer, boiled at
99C for 5min, incubated at 45C for 5min and centrifuged for 5min. Samples were washed,
stained and scanned using the Genechip Scanner 3000 7G and integrated command console
software. Total miR was isolated using the RNeasy mini kit (Qiagen, Germantown, MD) and
RT-PCR performed with the All-in-One™ miR qRT-PCR kits and validated primers
(GeneCopoeia, Rockville, MD) in the CFX96 RT-PCR detection system (Biorad, Hercules, CA).
∆∆
Calculations
for
expression
levels
were
made
using
the
Ct
method.
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Supplementary Table 2. Northern Blot probe sequences
miRNA
miR-29b
U6 snRNA
Lysine tRNA
Probe Sequence (5’ – 3’)
AACACTGATTTCAAATGGTGCTA
CACGAATTTGCGTGTCATCCTT
TACCGACTGAGCTATCCGGGC
Supplementary Table 2. Transfection efficiency was verified by probing the subcellular
fractions by northern blot with tRNAlys (cytoplasmic fraction) or U6 snRNA (nuclear fraction).
Total RNA (10ug) was harvested using PARIS™ Kit (Invitrogen) and was separated on
formaldehyde-agarose gels and transferred to Hybond™ nucleic acid blotting membrane (GE
Healthcare, Buckinghamshire, England). Oligonucleotide probes were 5' end-labeled with
digoxigenin-3-0-methylcarbonyl-ε-aminocaproic acid-N-hydroxy-succinimide ester (DIG).
Hybridizations and washes were performed at 52oC and 65oC respectively in a model 400
hybridization oven (Robbin’s Scientific, Sunnyvale, CA). Blots were incubated with antidigoxigenin-AP, Fab fragments (Roche Diagnostics, Indianapolis, IN) followed by CSPD readyto-use (Roche). Chemiluminescence was visualized on Hyblot CL® Autorad film (Denville
Scientific, NJ) using a M35A X-OMAT X-Ray film processor (Eastman Kodak, Rochester, NY).
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Supplementary Table 3. Proteasome genes upregulated in bortezomib-resistant cells
Gene
Fold change
Log-fold
PSMA1 proteasome subunit, alpha type, 1
7.3
2.9
PSME3 proteasome activator subunit 3 (PA28 gamma)
3.5
1.8
PSMD1 proteasome 26S subunit, non-ATPase, 1
3.2
1.8
PSMD2 proteasome 26S subunit, non-ATPase, 2
3.1
1.6
PSME3 proteasome activator subunit 3 (PA28 gamma
2.9
1.5
PSMD2 proteasome 26S subunit, non-ATPase
2.9
1.5
PSME3 proteasome activator subunit 3 (PA28 gamma)
2.8
1.5
PSMD1 proteasome 26S subunit, non-ATPase 1
2.7
1.4
PSME4 proteasome activator subunit 4 (PA200)
2.5
1.4
PSMF1 proteasome inhibitor subunit 1
2.3
1.2
PSMD2 proteasome 26S subunit, non-ATPase, 2
2.2
1.2
PSMA3 proteasome subunit, alpha type, 3
2.2
1.2
PSMC3IP PSMC3 interacting protein
2.2
1.1
PSMC3 proteasome 26S subunit, ATPase, 3
2.1
1.0
PSME2 proteasome activator subunit 3 (PA28 gamma)
2.0
1.0
PSMC2 proteasome 26S subunit, ATPase, 2
2.0
1.0
PSMB2 subunit, beta type, 2
2.0
1.0
PSMD12 proteasome 26S subunit, non-ATPase 12
2.0
1.0
PSMD9 proteasome 26S subunit non-ATPase 9
1.9
1.0
PSMC4 proteasome 26S subunit ATPase 4
1.8
0.9
PSMG1 proteasome assembly chaperone 1
1.8
0.8
PSMA4 proteasome subunit alpha type 4
1.8
0.8
PSMC5 proteasome 26S subunit ATPase 5
1.8
0.8
PSMA3 proteasome subunit alpha type 3
1.7
0.8
PSMB3 proteasome subunit beta type 3
1.7
0.8
PSMB6 proteasome subunit beta type 6
1.7
0.7
PSMD3 proteasome 26S subunit, non-ATPase 3
1.7
0.7
PSMA7 proteasome subunit alpha type 7
1.6
0.7
PSMD11 proteasome 26S subunit, non-ATPase 11
1.6
0.7
PSMD12 proteasome 26S subunit , non-ATPase 12
1.6
0.7
PSMC2 proteasome 26S subunit, ATPase 2
1.6
0.6
Autophagy Pathway genes upregulated in bortezomib-resistant cells
Gene
Fold change
ATG3 autophagy related 3 homolog
6.6
ATG5 autophagy related 5 homolog
1.8
ATG13 autophagy related 13 homolog
1.7
ATG4D autophagy related 4D homolog
1.6
ATG4 autophagy related 4 homolog
1.6
Log-fold
2.7
0.8
0.7
0.7
0.7
Autophagy Pathway genes downregulated in bortezomib-resistant cells
Gene
Fold change
ATG14 autophagy related 14 homolog
6.8
Log-fold
2.7
3
ATG9B autophagy related 9B homolog
1.9
0.7
Supplementary Table 3.
Gene Expression Microarray: Total mRNA was isolated from parental and resistant cells using
the RNeasy kit (Qiagen, Inc., Germantown, MD). Samples were analyzed using the Genechip
primeview assay for target synthesis and labeling. Samples were hybridized to the Genechip
primeview human gene expression array and those demonstrating a cutoff greater or less than
1.5-fold difference from the parental cell line used for further analysis. The quality of the total
RNA was confirmed using the Agilent 2100 Bioanalyzer using the RNA 6000 Nano Assay. For
each sample, the 3’ IVT Express Kit (Affymetrix, Santa Clara, CA) synthesizes biotin – label
cRNA target from 100ng of total RNA. A hybridization cocktail whic
target is created for each sample. The samples are hybridized to a standard Probe Array Cartidge
(GeneChip Primeview Human Gene Expression Array–Affymetrix) in the GeneChip
Hybridization Oven 640 (Affymetrix). Probe arrays are washed and stained using the Fluidics
Station 450 (Affymetrix). Arrays were scanned with the Affymetrix GeneChip Scanner 3000 7G.
Raw data files were created by Command Console, the Affymetrix operating software program.
The Affymetrix Expression Console Program was used to examine the Affymetrix Gene Array
quality control factors for all samples in a project. Two separate independent algorithms were
used to normalize the data for all samples within a project. Data is normalized using the Robust
Multi-Array Analysis (RMA) algorithm and the MAS5 algorithm. Specific quality control
metrics examined include signal histogram, relative log expression signal, Pearson's correlation,
pm mean (average signal intensity of probes), % genes called present, 3’5’ ratio of housekeeping
genes, expression values of spiked-in poly-A RNA controls, and values of spiked-in
hybridization controls.
Software Programs: The quality of total RNA was checked by the Agilent 2100 Bioanalyzer
using the RNA 6000 Nano Assay. For each sample, the 3’ IVT Express Kit (Affymetrix)
synthesizes biotin – label cRNA target from 100ng of total RNA. A hybridization cocktail which
includes 10 micrograms of cRNA target is created for each sample. The samples are hybridized
to a standard Probe Array Cartidge (GeneChip Primeview Human Gene Expression Array–
Affymetrix) in the GeneChip Hybridization Oven 640 (Affymetrix). Probe arrays are washed and
stained using the Fluidics Station 450 (Affymetrix). The arrays are scanned with the Affymetrix
GeneChip Scanner 3000 7G. Raw data files are created by Command Console, the Affymetrix
operating software program. The Affymetrix Expression Console Program is used to examine the
Affymetrix Gene Array quality control factors for all samples in a project. Two separate
independent algorithms are used to normalize the data for all samples within a project. Data is
normalized using the Robust Multi-Array Analysis (RMA) algorithm and the MAS5 algorithm.
Specific quality control metrics examined include signal histogram, relative log expression
signal, Pearson's correlation, pm mean (average signal intensity of probes), % genes called
present, 3’5’ ratio of housekeeping genes, expression values of spiked-in poly-A RNA controls,
and values of spiked-in hybridization controls.
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Supplementary Figure 1. Dose-dependent effect of PIs on parental cells and PI-resistant cells.
BTZ-Resistant
Bortezomib (nM)
CFZ-Resistant
Carfilzomib (nM)
IXZ-Resistant
Ixazomib (nM)
Supplementary Figure 1. Parental (drug-naïve) RPMI8226 cells or RPMI8226 cells that were generated separately with acquired
resistance to the PIs bortezomib, carfilzomib or ixazomib (MLN9708) were treated with PIs at indicated concentrations. Cells were
then cultured for 72h under standard conditions and viability determined using the XTT assay. Shown is the average of triplicate
measurements.
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Supplementary Figure 2. MiR replacement effect on MM, lymphoma, amyloidosis and Waldenströms cells.
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Supplementary Figure 2. Myeloma cells KMS28BM, MM1.S, the lymphoma cell line KG-1, amyloidosis cell lines AMCL-1 and
AMCL-2 and the Waldenström’s cell line WMCL-1 were transfected with indicated miR replacements, cultured for 72h, treated with
bortezomib and viability determined using the XTT assay. Shown are the average of triplicate measurements.
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