Caracterización de la asignatura Física en primer semestre de FOC

There were 21 putative hits for the U2OS minus arsenite samples (Table 4.4B), the top ten of which by p-value are presented in Table 4.4A. There were 76 putative hits for the U2OS plus arsenite samples (Table 4.4D), of which the top ten by p-value are tabulate below (Table 4.4C). There were 9 proteins that were putative hits under both conditions (Table 4.4E).

Table 4.4A: Top ten hits U2OS minus arsenite, by t-test p-value

Protein U2OS1 U2OS2 U2OS3 U2OS4 U2OS5 U2OS6 p-value

EWSR1 220690 79529 100240 316360 171140 180290 0.0027 VIM 102930 164410 127120 138130 192810 160180 0.0039 LDB1 0 0 0 245600 208200 300600 0.0112 RPS27 64800 111000 122000 104000 165000 190000 0.0234 UNR 0 0 0 644760 328380 580240 0.033 ACTN1 0 367530 0 85461 453590 45127 0.0334 DDX3X 591330 228460 573350 970920 411940 933630 0.0387 TMEM97 0 0 0 87815 59805 128300 0.0437 ATP5A1 151760 872820 76693 1127100 1344900 683320 0.045 RPS7 189240 0 0 224140 55669 79267 0.0476 HSPA5 0 449500 0 51703 578090 99651 0.0532

N.B. IgG samples shaded lilac repeat 1-3 running left to right (U2OS1-U2OS3). The corresponding UNR samples are shaded green and also run left to right (U2OS4-U2OS6). The t-test p-values are for a two tailed paired t-test without multiple testing correction. Yellow shading denotes proteins that were recorded in UNR samples only.

Table 4.4B: Putative UNR-interacting proteins from unstressed U2OS cells, by t-test p-value

Protein p-value

Cluster of B0QYK0_HUMAN RNA-binding protein EWS (GN=EWSR1) 0.0027

Cluster of VIME_HUMAN Vimentin (GN=VIM) 0.0039

Cluster of LDB1_HUMAN LIM domain-binding protein 1 (GN=LDB1) 0.0112

RS27_HUMAN 40S ribosomal protein S27 (GN=RPS27) 0.0234

CSDE1_HUMAN Isoform 4 of Cold shock domain-containing protein E1

(GN=CSDE1) 0.0330

Cluster of ACTN1_HUMAN Alpha-actinin-1 (GN=ACTN1) 0.0334

Cluster of A0A0D9SF53_HUMAN ATP-dependent RNA helicase DDX3X

(GN=DDX3X) 0.0387

TMM97_HUMAN Transmembrane protein 97 (GN=TMEM97) 0.0437

ATPA_HUMAN ATP synthase subunit alpha, mitochondrial (GN=ATP5A1) 0.0450

RS7_HUMAN 40S ribosomal protein S7 (GN=RPS7) 0.0476

GRP78_HUMAN 78 kDa glucose-regulated protein (GN=HSPA5) 0.0532 Cluster of SSBP3_HUMAN Single-stranded DNA-binding protein 3 (GN=SSBP3) 0.0544 Cluster of HUWE1_HUMAN Isoform 3 of E3 ubiquitin-protein ligase HUWE1

(GN=HUWE1) 0.0686

RS8_HUMAN 40S ribosomal protein S8 (GN=RPS8) 0.0768

SRSF2_HUMAN Serine/arginine-rich splicing factor 2 (GN=SRSF2) 0.0812

RL29_HUMAN 60S ribosomal protein L29 (GN=RPL29) 0.0845

H4_HUMAN Histone H4 (GN=HIST1H4A) 0.0850

STRAP_HUMAN Serine-threonine kinase receptor-associated protein (GN=STRAP) 0.1160 E9PK91_HUMAN Bcl-2-associated transcription factor 1 (GN=BCLAF1) 0.1264 NARR_HUMAN Ras-related protein RAB34, isoform NARR (GN=RAB34) 0.1670 U2AF2_HUMAN Splicing factor U2AF 65 kDa subunit (GN=U2AF2) 0.1769 N.B. The t-test p-values are for a two tailed paired t-test without multiple testing correction. Yellow shading denotes proteins that were recorded in UNR samples only.

Table 4.4C: Top ten hits U2OS plus arsenite, by t-test p-value

Protein U2OS7 U2OS8 U2OS9 U2OS10 U2OS11 U2OS12 p-value

ANXA2 0 0 0 91468 90853 81731 0.0013 UNRIP 0 0 0 3243000 3005700 3508700 0.002 UNR 0 0 0 3774400 3703900 4439600 0.0035 HSPA9 0 0 0 161500 127070 186940 0.0118 RPL11 35120 59251 118000 102600 157590 193990 0.0128 TUBB6 0 85370 347680 626070 893540 1367000 0.0188 LDB1 0 0 0 285440 209560 356440 0.0216 RPS2 332580 172460 302870 733490 874400 880930 0.0235 NARR 0 0 0 282330 159030 290080 0.029 RBM14 0 0 0 105720 72114 138340 0.0314 TUBB 407100 1326300 1790500 4629400 3511500 4856900 0.0332

N.B. IgG samples shaded lilac repeat 1-3 running left to right (U2OS7-U2OS9). The corresponding UNR samples are shaded green and also run left to right (U2OS10-U2OS12). The t-test p-values are for a two tailed paired t-test without multiple testing correction. Yellow shading denotes proteins that were recorded in UNR samples only.

Table 4.4D: Putative UNR-interacting proteins from arsenite stressed U2OS cells, by t-test p-value

Protein p-value

Cluster of ANXA2_HUMAN Annexin A2 (GN=ANXA2) 0.0013

STRAP_HUMAN Serine-threonine kinase receptor-associated protein (GN=STRAP) 0.0020 CSDE1_HUMAN Isoform 4 of Cold shock domain-containing protein E1

(GN=CSDE1) 0.0035

Cluster of GRP75_HUMAN Stress-70 protein, mitochondrial (GN=HSPA9) 0.0118

RL11_HUMAN 60S ribosomal protein L11 (GN=RPL11) 0.0128

TBB6_HUMAN Tubulin beta-6 chain (GN=TUBB6) 0.0188

Cluster of LDB1_HUMAN LIM domain-binding protein 1 (GN=LDB1) 0.0216 Cluster of RS2_HUMAN 40S ribosomal protein S2 (GN=RPS2) 0.0235 NARR_HUMAN Ras-related protein RAB34, isoform NARR (GN=RAB34) 0.0290 Cluster of RBM14_HUMAN RNA-binding protein 14 (GN=RBM14) 0.0314

Cluster of TBB5_HUMAN Tubulin beta chain (GN=TUBB) 0.0332

Cluster of C9JJQ8_HUMAN Tubulin alpha-4A chain (Fragment) (GN=TUBA4A) 0.0344 A0A087WXM6_HUMAN 60S ribosomal protein L17 (Fragment) (GN=RPL17) 0.0348 Cluster of RINI_HUMAN Ribonuclease inhibitor (GN=RNH1) 0.0371 Cluster of HUWE1_HUMAN Isoform 3 of E3 ubiquitin-protein ligase HUWE1

(GN=HUWE1) 0.0400

RL8_HUMAN 60S ribosomal protein L8 (GN=RPL8) 0.0444

Cluster of HS71A_HUMAN Heat shock 70 kDa protein 1A (GN=HSPA1A) 0.0461 Cluster of B5MDF5_HUMAN GTP-binding nuclear protein Ran (GN=RAN) 0.0468 Cluster of J3KQ22_HUMAN Pyrroline-5-carboxylate reductase (Fragment)

(GN=PYCR1) 0.0510

HSPB1_HUMAN Heat shock protein beta-1 (GN=HSPB1) 0.0526

Cluster of RS30_HUMAN 40S ribosomal protein S30 (GN=FAU) 0.0561 DYHC1_HUMAN Cytoplasmic dynein 1 heavy chain 1 (GN=DYNC1H1) 0.0563 Cluster of SSBP3_HUMAN Single-stranded DNA-binding protein 3 (GN=SSBP3) 0.0565 AHNK_HUMAN Neuroblast differentiation-associated protein AHNAK

(GN=AHNAK) 0.0567

RS8_HUMAN 40S ribosomal protein S8 (GN=RPS8) 0.0580

MCM7_HUMAN DNA replication licensing factor MCM7 (GN=MCM7) 0.0634

Cluster of FLNC_HUMAN Filamin-C (GN=FLNC) 0.0650

Cluster of H2B1A_HUMAN Histone H2B type 1-A (GN=HIST1H2BA) 0.0655 Cluster of H0Y449_HUMAN Nuclease-sensitive element-binding protein 1

(Fragment) (GN=YBX1) 0.0681

Cluster of RS3_HUMAN 40S ribosomal protein S3 (GN=RPS3) 0.0682 Cluster of H7C1H2_HUMAN 26S proteasome non-ATPase regulatory subunit 2

(Fragment) (GN=PSMD2) 0.0686

Cluster of RS9_HUMAN 40S ribosomal protein S9 (GN=RPS9) 0.0704 Cluster of BAG2_HUMAN BAG family molecular chaperone regulator 2

(GN=BAG2) 0.0731

Cluster of HNRPK_HUMAN Heterogeneous nuclear ribonucleoprotein K

(GN=HNRNPK) 0.0757

K7EJV9_HUMAN 60S ribosomal protein L23a (Fragment) (GN=RPL23A) 0.0811

Cluster of E9PFH4_HUMAN Transportin-3 (GN=TNPO3) 0.0824

Cluster of TFG_HUMAN Protein TFG (GN=TFG) 0.0835

ATPA_HUMAN ATP synthase subunit alpha, mitochondrial (GN=ATP5A1) 0.0848 MSH6_HUMAN DNA mismatch repair protein Msh6 (GN=MSH6) 0.0887 Cluster of PCBP1_HUMAN Poly(rC)-binding protein 1 (GN=PCBP1) 0.0887

RS14_HUMAN 40S ribosomal protein S14 (GN=RPS14) 0.0916

Cluster of ADT2_HUMAN ADP/ATP translocase 2 (GN=SLC25A5) 0.0986

EMD_HUMAN Emerin (GN=EMD) 0.1043

Cluster of A0A0A0MSG2_HUMAN Four and a half LIM domains protein 2

(GN=FHL2) 0.1119

Cluster of COF1_HUMAN Cofilin-1 (GN=CFL1) 0.1119

Cluster of PLEC_HUMAN Plectin (GN=PLEC) 0.1119

Cluster of SQSTM_HUMAN Sequestosome-1 (GN=SQSTM1) 0.1147

Cluster of TRIP6_HUMAN Thyroid receptor-interacting protein 6 (GN=TRIP6) 0.1148

Cluster of IPO4_HUMAN Importin-4 (GN=IPO4) 0.1188

SSRD_HUMAN Translocon-associated protein subunit delta (GN=SSR4) 0.1205 Cluster of TIF1B_HUMAN Transcription intermediary factor 1-beta (GN=TRIM28) 0.1224 U2AF2_HUMAN Splicing factor U2AF 65 kDa subunit (GN=U2AF2) 0.1312 Cluster of ATPO_HUMAN ATP synthase subunit O, mitochondrial (GN=ATP5O) 0.1335 Cluster of MPCP_HUMAN Phosphate carrier protein, mitochondrial

(GN=SLC25A3) 0.1414

Cluster of M0QXS5_HUMAN Heterogeneous nuclear ribonucleoprotein L

(Fragment) (GN=HNRNPL) 0.1428

Cluster of J3QL54_HUMAN Nuclear pore complex protein Nup85 (GN=NUP85) 0.1526 Cluster of F8W6I7_HUMAN Heterogeneous nuclear ribonucleoprotein A1

(GN=HNRNPA1) 0.1541

Cluster of H3BVG0_HUMAN Nuclear pore complex protein Nup93 (GN=NUP93) 0.1559 Cluster of Q5JR05_HUMAN Rho-related GTP-binding protein RhoC (GN=RHOC) 0.1593 Cluster of ATD3A_HUMAN Isoform 2 of ATPase family AAA domain-containing

protein 3A (GN=ATAD3A) 0.1635

EF2_HUMAN Elongation factor 2 (GN=EEF2) 0.1708

Cluster of RL28_HUMAN 60S ribosomal protein L28 (GN=RPL28) 0.1798 Cluster of E9PR16_HUMAN Nuclear pore complex protein Nup160 (Fragment)

(GN=NUP160) 0.1936

Cluster of 1433B_HUMAN 14-3-3 protein beta/alpha (GN=YWHAB) 0.1958 Cluster of IMB1_HUMAN Importin subunit beta-1 (GN=KPNB1) 0.2152 Cluster of H7C2U6_HUMAN Protein NipSnap homolog 1 (Fragment)

(GN=NIPSNAP1) 0.2295

Cluster of C9JZR2_HUMAN Catenin delta-1 (GN=CTNND1) 0.2330 Cluster of B4DR61_HUMAN Protein transport protein Sec61 subunit alpha

isoform 1 (GN=SEC61A1) 0.2499

Cluster of RBP2_HUMAN E3 SUMO-protein ligase RanBP2 (GN=RANBP2) 0.2538

Cluster of PLAP_HUMAN Phospholipase A-2-activating protein (GN=PLAA) 0.2931 A0A0A0MSX9_HUMAN Isoleucine--tRNA ligase, cytoplasmic (GN=IARS) 0.3092 Cluster of Q5SZU1_HUMAN D-3-phosphoglycerate dehydrogenase (GN=PHGDH) 0.3112 SYEP_HUMAN Bifunctional glutamate/proline--tRNA ligase (GN=EPRS) 0.3125

MVP_HUMAN Major vault protein (GN=MVP) 0.3478

N.B. The t-test p-values are for a two tailed paired t-test without multiple testing correction. Yellow shading denotes proteins that were recorded in UNR samples only.

Table 4.4E: Putative UNR interacting proteins in both plus and minus arsenite U2OS samples

STRAP_HUMAN Serine-threonine kinase receptor-associated protein (GN=STRAP) CSDE1_HUMAN Isoform 4 of Cold shock domain-containing protein E1 (GN=CSDE1) Cluster of LDB1_HUMAN LIM domain-binding protein 1 (GN=LDB1)

NARR_HUMAN Ras-related protein RAB34, isoform NARR (GN=RAB34)

Cluster of HUWE1_HUMAN Isoform 3 of E3 ubiquitin-protein ligase HUWE1 (GN=HUWE1) Cluster of SSBP3_HUMAN Single-stranded DNA-binding protein 3 (GN=SSBP3)

RS8_HUMAN 40S ribosomal protein S8 (GN=RPS8)

ATPA_HUMAN ATP synthase subunit alpha, mitochondrial (GN=ATP5A1) U2AF2_HUMAN Splicing factor U2AF 65 kDa subunit (GN=U2AF2)

At first sight, there appeared to be several recurrent proteins that could be true UNR-interactors. UNRIP, LDB1, NARR and HUWE1 were considered putative hits from both the plus and minus arsenite U2OS samples (Table 4.4E) and had also been putative hits from the plus and minus arsenite HeLa samples (Table 4.3). On top of this, it was noted that SSBP3 was a putative UNR- interactor in stressed and unstressed U2OS cells (Table 4.4E). This had also been a hit in both of the trial runs that used unstressed HeLa cells (Table 4.1).

Among other observations, it was noted that 78 kDa glucose-regulated protein (HSPA5) was a hit for the minus arsenite samples, as it was for the HeLa gel slice trial run (Figure 4.12). Also, SQSTM1 was present in the list of putative hits for U2OS plus arsenite, as was DYNC1H1, the top hit for HeLa plus arsenite. See the Progenesis/quantitative analysis section below for further consideration of the similarities and differences between the cell types.

Having had an initial look at the U2OS data, it was decided to have a similar look at the SaOS-2 data prior to moving on to analyse the main data further using Progenesis.