52 patients in the TCGA cohort. Yuqian Yan et al. AKT and AR ... An illustration shows the workflow of a 3D Matrigel culture system. D, E C4-2 cells were infected ...
Data information: Representative images are shown only for SOD1/IKK. ... phase of ALS disease (based on peak weight); (MantelâCox **P = 0.0048). ... G Quantification of IBA1+ cells of C-59- or Veh-treated mice from P90 to P102.
catalytically inactive Cas9 fused to the transcriptional repressor Mxi1 (dCas9-Mxi1), and a guide RNA targeting the RBD2 gene locus. Data were normalized ...
Tuba1a. Error bars, standard error of the mean (s.e.m.); n = 3. B The two transcript isoforms of Gata6 and Nkx2-1 are differentially regulated during lung cancer ...
immunofluorescence (IF) analysis with ubiquitin (FK2) antibody. B .... F Co-IP analysis of the interaction between TRIM16 and NRF2 in HEK293T lysates of cells ...
with pcDNA3 or with a plasmid for the overexpression of ATPIF1. The results are representative of three independent experiments. Scale bar = 10 lm.
A Sagittal T1-weighted FSE showing thinning of the posterior part of the corpus callosum with agenesis of the splenium. Prominent subcutaneous fat deposition ...
Figure EV1. Blood vessel dysmorphia arises during glioma growth. A HIF1o immunohistochemistry on 5-week growth glioma in ROSAmTmG::Pdgfb-iCre mouse ...
347.6; Rab7A Ð332.4 to 357.2; Rab8A Ð378.6 to 311.0; Rab8B Ð346.7 to 342.9; Rab10 Ð372.3 to 317.3; Rab12 Ð499.8 to 189.8; Rab29 Ð865.3 to Ð175.7; ...
Expanded View Figures. â¸. Figure EV1. SOX2 marks a subset of acinar cells that replenish acini. A Wild-type murine PG stained for SOX2, ECAD, and nuclei.
E, F Effects of cisplatin (E) and C18-pyr-cer (F) on cell death (48 h) in UM-SCC-22A [HPV(Ã)] versus UM-SCC-47 [HPV(+)] cells were measured by trypan blue ...
Figure EV1. Characterization of the transcriptional responses in HSPC. A. Principal component analysis (PCA) plot. The samples from the RNA-seq experiment ...
Figure EV1. Cdc14 is required for recombinational DNA repair. A Constitutive expression of HO produces a constant DSB at the MAT locus in wild-type and ...
Figure EV1. Mapping of chemical shift perturbations occurring upon the formation of ... dsRBD12, are shown in blue, red, and black, ... Unmatched black peaks.
Expanded View Figures. â¸. Figure EV1. Mcp1 interacts with Klp5/Klp6 and requires its motor activity to accumulate at microtubule plus ends but is dispensable ...
LRP6 possesses four YWTD domains where the second (E2) and the fourth (E4) YWTD domains constitute the EGF-YWTD-EGF units. The N-terminal.
The yellow line indicates the edge of Pxn-GFP (green)-positive areas at 0 min, and the blue line indicates the edge at the end of 30 ... Samuel J Atkinson et al.
Figure EV1. ZBTB48 ZnF11 is necessary to bind to telomeres. A Sequence-specific DNA pull-downs with either telomeric (TTAGGG) or a control sequence ...
Expanded View Figures. A. B. Figure EV1. Insufficient anchoring, gel polymerization, or digestion can affect the sample integrity. A An intact sample of COS7 cell ...
Figure EV1. Inhibition of USP7 and USP47 blocks NLRP3 inflammasome activation in mouse BMDMs. A IL-1b ELISA in supernatants of LPS-primed (1 lg/ml, ...
F Cell viability measurements à doxycycline (72â96 h) for the non-targeting scrambled shRNA (NT CTL), CDK2, and MITF conditional knockdown cell lines in ...
Alireza Azimi et al
Molecular Systems Biology
CDK2i overcomes BRAFi-Hsp90i resistance
Expanded View Figures Only for b
A Heating T (˚C)
TPP
Centrifugation and recover supernatant
TMT Labelling
Melt curve
TMT 2
...
...
DMSO
m/z
1:1 (v:v)
Phospho-TPP
TMT ... Melt curve
For each protein: TMT ...
T10
TiO2 enrichment
TMT 10
m/z
B
C
Melting curves of the same protein +/- drug Fraction non-denatured
+DMSO + drug
T (˚C)
‘Intact cells’ layout (in vivo conditions)
Target protein that is stabilized by the interaction with the drug
Primary targets
SK-Mel 24 Drug / DMSO ‘Intact cells’ 37
Primary & secondary (downstream) targets
SK-Mel 24 - Intact cells 37
40 43
46
49 67 70
52
55 58
5
SK-Mel 28 Drug / DMSO ‘Intact cells’
SK-Mel 24 Drug / DMSO ‘Intact cells’ 37
45
5
34
SK-Mel 24 Drug / DMSO ’Lysate’
SK-Mel 28 Drug / DMSO ’Lysate’ 7
T (˚C)
41
30
SK-Mel 28 Drug / DMSO ’Lysate’ 20
28
SK-Mel 28 - Intact cells 61
64
- + - + - + - + - + - + - + - + - + - + - + - +
90
SK-Mel 24 Drug / DMSO ’Lysate’
SK-Mel 28 Drug / DMSO ‘Intact cells’ 32
E kDa
D
‘Lysate’ layout (in vitro conditions)
T (˚C)
TMT 3
Fraction non-denatured
b ‘Intact cells’ layout Intact cells (NOT Treatment lysates!) are harvested with with and aliquoted drug /
bRP/ LC/MS/MS
Intensity
T3
OR
Intensity
T2
Protein digestion
a
TMT 1
Cooling
Cell lysis: freeze and thaw
T1
Cell lysis by freeze & thaw
Lysate incubation with +Drug / DMSO
Fraction non-denatured
For each protein:
‘Lysate’ layout
T (˚C) XL888 Hsp90α and β
37 kDa 90
40 43
46
49 67 70
52
55 58
61
64
- + - + - + - + - + - + - + - + - + - + - + - +
T (˚C) XL888 Hsp90α and β
44
CDC37
44
CDC37
42
β-actin
42
β-actin
F
Lysate / intact cells Cell line 2 (+DMSO (or drug))
Melting curves of the same protein in different cell lines Protein that is more stable in Cell line 1 cell line 2 due to Cell line 2 a different PTMs status / mutation etc. Fraction non-denatured
Lysate / intact cells Cell line 1 (+DMSO (or drug))
‘Meltome’ of resistant / sensitive cell lines
T (˚C)
Figure EV1.
ª 2018 The Authors
Molecular Systems Biology 14: e7858 | 2018
EV1
Molecular Systems Biology
◀
CDK2i overcomes BRAFi-Hsp90i resistance
Alireza Azimi et al
Figure EV1. TPP platform employed in this study. A Workflow of the TPP platforms (“lysate” and “intact cells”) used to measure the proteome and phosphoproteome thermal stability upon drug treatment using a TMT approach. B The comparison of the proteome thermal stability upon different conditions (+/ drug) enables to identify the drug targets. C The two layouts “lysate” and “intact cells” provide complementary information regarding the nature of the targets (primary drug targets or secondary targets; Franken et al, 2015). D Venn diagrams of the entries retrieved by the comparisons in different settings. Experiments were performed in two biological replicates. E Validation of Hsp90 and CDC37 thermal shift by Western blot. F The TPP workflow enables the comparison of the thermal stability of proteome and phosphoproteome of resistant versus sensitive cells to the Hsp90i XL888.
Log2 ratio fold change Protein Phosphosite: activity ON
-5.4 deStabilized
0
12.4 Stabilized
Phosphosite: activity OFF Phosphosite: other functions Hsp90 interactor
Figure EV2. TPP and phospho-TPP protein interaction maps in sensitive and resistant cells. Protein interaction map built using Cytoscape 3.2 and Reactome as plugin (see Appendix) and the statistical significant entries generated from the comparison of the proteome and phosphoproteome of drug versus DMSO for sensitive and resistant cells in both “lysate” and “intact cell” layouts.
APEH PARP4 GEMIN6 SHMT2 RNF141 MYOM1 1 DHTKD1 KD1 D CPOX OX XM MMAB PCMT1 T1 POP4 CHURC1 PGM2L1 HERC2 PEBP1 APRT FASTKD5 WDR92 XL888 CRKLPMM2 NDUFB3 N KIF7 C11orf98 C1QBP MCOLN2 TXNDC5 FABP7 NFRKB SEPT6 PT T6 T SARS2 TTC39C APOA1BP PGM1 CTSL SF LRASSF3 MRTO4 4 ELF1 E DPYSL2 D 2 JMJD7 PDE5A ATPAF2 ALDOA HSPD1 CLIP4 C CD2BP2 CDK6 C GTPBP3 SF3B2 CDK4 CYB5R2 TUBGCP6 RACGAP1
APRT
CNDP2
DYNC1LI1 I1
Alireza Azimi et al
CRK
ACOT13
NAPA PDE5A
SRC
CA13 XL888
HSPD1 FASTKD5
DIP2B CDK6 ACP2
PKM PAK2 ARFGAP2 SDHB
PPP3CB C CDK4 CDK2 RRP9
CNDP2 PARP4 ACP1 RACGAP1 MSANTD4 GLUD2 CRKL PGM5 GLDC G DYNC1H1 1 PCMT1 ITSN2 CFL2 HSCB DHTKD1 H D DH PEPD RAD21
GSTZ1
DYNC1H1
Log2 ratio fold change Protein Phosphosite: activity ON
-8.9
0
deStabilized
Phosphosite: activity OFF
16.9 Stabilized
Phosphosite: other functions Hsp90 interactor
Figure EV3. Proteome thermal stability of resistant versus sensitive cells. Protein interaction map built using Cytoscape 3.2 and Reactome as plugin (see Appendix) and the statistical significant entries generated from the comparison of the proteome and phosphoproteome thermal stability of resistant versus sensitive cells different settings.
EV4
Molecular Systems Biology
14: e7858 | 2018
ª 2018 The Authors
Alireza Azimi et al
CDK2i overcomes BRAFi-Hsp90i resistance
Molecular Systems Biology
Figure EV4. Validation of the TPP and proteomics findings. A B C D E F
Melting curves of some hits generated by the comparison of the baseline thermal stability of SK-Mel 24 and SK-Mel 28 in lysate and intact cell settings. Validation of pPAK4 thermal shift by Western blot. Principal component analysis (PCA) of the proteomics (left panel) and phosphoproteomics (right panel) results in different settings. Western blot analyses of SK-Mel 24 and SK-Mel 28 upon BRAFi treatment show phosphorylation and activation of pERK. Protein expression levels of the shared kinases statistically significant regulated in SK-Mel 24 and SK-Mel 28 upon treatment with BRAFi-Hsp90i/DMSO. Cell viability measurements doxycycline (72–96 h) for the non-targeting scrambled shRNA (NT CTL), CDK2, and MITF conditional knockdown cell lines in DMSO (SD is plotted; n = 3). G Western blots of protein expression levels of CDK2 in A375 DR1, ESTDAB 37, M026.X1.CL, and MNT-1 DR100 upon treatment with DMSO, BRAFi, and Hsp90i at 72 h (left panel). Band intensities for the quantification of CDK2 expression levels in different cell lines in different settings were normalized against the mean of GAPDH, and DMSO treatment was used as reference (right panel). H Western blot of protein expression levels of AKT1 in SK-Mel 24 and SK-Mel 28 in different settings at 48-h treatment.
