(A) CKS1B signal for 51 paired arrays was obtained at diagnosis and relapse. The high risk (quartile 4) reference line is taken from the complete (n=351) sample of arrays at diagnosis. Note that a majority of samples showed increased expression at relapse; the most dramatic changes were observed in patients with expression levels in quartiles 1–3 at diagnosis. A paired Student t test was used to compare log-scale signal at diagnosis and relapse. (B) Kaplan-Meier analysis of postrelapse survival is shown in relation to CKS1B expression from low expression at baseline (BL-Low) to low expression at relapse (RL-Low; n = 15) and BL-Low to high expression at relapse (RL-High; n = 23) and already high expression at baseline (BL-High; n = 13) determined by microarray. At the time of analysis, the median follow-up of a post-relapse survival was 14 months (range, 0.3 to 50 months) in this analysis.

CKS1B was knocked down in KMS28PE, OCI-MY5 and XG-1 cells. Cells were cultured for 72 hours and cell lysates were prepared. (A) CKS1B-induced signaling pathways were screened by Western blots in CKS1B-knockdown (CKS1B-sh) OCI-MY5 cells using the indicated antibodies. (B) Protein levels of p-STAT3, p-MEK1/2, p-ERK1/2 and p-BCL2 were examined in KMS28PE, OCI-MY5 and XG1 cells after CKS1B-knockdown. Wild-type (WT) and Scramble (SCR)-transfected cells were used as controls and β-actin was used as loading control. (C–E) Protein levels of p-STAT3, p-MEK1/2, p-ERK1/2 and p-BCL2 were examined by Western blot analysis in CKS1B-transfected, SKP2-silenced, and p27 ^Kip1-transfected MM cell lines of OCI-MY5 and XG-1 cells, respectively. WT and EV cells were used as controls and β-actin was used as loading control.

(A) Western blots showed that MEK inhibitor U0126 inhibited p-MEK, p-ERK, and p-BCL2 expression. 1×10⁶CKS1B -transfected OCI -MY5 and XG-1 were treated with 10nM U0126 for 48 hours, and p-MEK1/2, p-ERK1/2 and p-BCL2 levels were analyzed. Untreated and EV cells with or without U0126 treatment were used as controls, and β-actin was used as loading control. (B) Cell growth and (C) cell death were evaluated. MM cells treated with U0126 showed clearly more cell growth inhibition (OCI-MY5: 2.84M vs. 2.01M, and XG1: 2.3M vs. 1.3M) and more cell death (OCI-MY5: 80% vs. 43%, and XG1: 77% vs. 52%) in CKS1B over-expressed cells than in empty vector (EV)-transfected controls. (D) Western blots showed p-BCL2 increased in MEK1 over-expressed MM cells. KMS28PE, OCI-MY5 and XG-1 were doubly transfected with CKS1B-shRNA and MEK1-cDNA to knockdown CKS1B and constitutively activate the MEK/ERK signaling pathway in these cells. SCR and CKS1B-sh cells were used as controls, and β-actin was used as loading control. (E) Cell growth and (F) cell death were evaluated in CKS1B-sh and MEK1 doubly-transfected cells after 72 hours induction. Clearly, MEK1 overexpression can partially rescue CKS1B-shRNA induced cell growth inhibition (KMS28PE: 1.74M vs. 3.10M, OCI-MY5: 2.10M vs. 3.63M, and XG1: 1.20M vs. 2.50M) and cell apoptosis (KMS28PE: 22% vs. 41%, OCI-MY5: 27% vs. 51%, and XG1: 33% vs. 56%). (G) Cell growth and (H) cell death were evaluated in the treatment of a specific BCL2 inhibitor (BCL2-inh). OCI-MY5 and XG-1 cells transfected with CKS1B showed a stronger cell growth inhibition (OCi-MY5: 1.97M vs. 0.85M, and XG1: 1.53M vs. 0.96M) and cell death (OCI-MY5: 60% vs. 17%, and XG1: 58% vs. 21%) compared with EV- transfected OCI-MY5 and XG-1. (I) Western blots showed BCL2 increased in OCI-MY5 and XG-1doubly transfected with CKS1B-shRNA and BCL2-cDNA. (J) Cell growth and (K) cell death were evaluated in CKS1B-sh and BCL2 doubly-transfected cells after 72 hours induction. Over-expression of BCL2 in MM cells can partially rescue CKS1B knockout induced cell growth inhibition (KMS28PE: 1.74M vs. 3.10M, OCI-MY5: 2.40M vs. 3.63M, and XG1: 2.60M vs. 4.20M) and apoptosis (KMS28PE: 32% vs. 55%, OCI-MY5: 43% vs. 59%, and XG1: 39% vs. 61%). Scramble (SCR) and CKS1B-sh cells served as controls. Results were expressed as Mean ±SD of three independent experiments ( *p <0 .05).

(A) Western blots examined CKS1B proteins increased in CKS1B-transfected OCI-MY5 and XG-1 cells (CKS1B-OE). (B - G) MM cells overexpressed CKS1B showed multi-drug resistance. CKS1B-over-expressing OCI-MY5 and XG-1 cells were treated with bortezomib (Vel; 5 nM), doxorubicin (Dox; 100 nM), and etoposide (Epo; 100 nM) for 48 hours in the cultures, and cell growth (B, OCI-MY5: 0.11M vs. 0.75M, and XG1: 0.06M vs. 0.65M; D, OCI:-MY5: 0.11M vs. 0.47M, and XG1: 0.19M vs. 0.54M; F, OCI-MY5: 0.21 vs. 0.39M, and XG1: 0.19 vs. 0.47M) and death (C,OCI-MY5: 34% vs. 63%, and XG1: 20% vs. 54%; E, OCI-MY5: 38% vs. 77%, and XG1: 53% vs. 81%; G, OCI-MY5: 41% vs. 71%, and XG1: 32% vs. 59%) were evaluated for these three treatments respectively. Untreated and EV cells with or without drug treatment were used as controls. Results were expressed as Mean+SD of three independent experiments (*p <0 .05).

(A) 1×10⁶ CKS1B- transfected OCI-MY5 and XG-1 were treated with 10nM Nifuroxazide (Nif) for 48 hours, and cell lysates were prepared. Protein levels of p-STAT3 and MCL1 were analyzed and showed decreased in Nif treated MM cells by Western blots. (B) Cell growth and (C) cell survival were evaluated. MM cells treated with Nif showed clearly more cell growth inhibition (OCI-MY5: 2.18M vs. 1.04M, and XG-1: 2.07M vs. 0.90M) and more cell death (OCI-MY5: 60% vs. 27%, and XG1: 58% vs. 31) in CKS1B over-expressed cells. Untreated and EV cells with or without Nif treatment were used as controls. (D) Western blots showed IGF-1 increased p-STAT3 and MCL1 in CKS1B silenced MM cells. 1×10⁶ OCI-MY5 and XG-1 after CKS1B-knockdown (CKS1B-sh) were treated with 100 ng/mL IGF-1 for 72 hours and p-STAT3 and MCL1 protein levels were analyzed. Untreated CKS1B-sh cells and SCR cells with or without IGF-1 treatment were used as controls. (E) Cell growth and (F) cell death were evaluated. IGF-1 can partially rescue CKS1B knockout induced cell growth inhibition (KMS28PE: 1.74M vs. 2.88M, OCI-MY5: 2.40M vs. 3.64M, and XG1: 2.60M vs. 3.86M) and apoptosis (KMS28PE: 36% vs. 56%, OCI-MY5: 43% vs. 61%, and XG1: 39% vs. 53%). Untreated cells and SCR cells with or without IGF-1 treatment were used as controls. Results were expressed as Mean ± SD of three independent experiments (*p <0 .05).

(A) Cell growth and (B) cell death were evaluated in CKS1B-transfected OCI-MY5 treated with MEK1 inhibitorU0126 (10 μM) plus STAT3 inhibitor Nifuroxazide (10 nM) (N+U Inh) and Nifuroxazide plus BCL2 inhibitor (10 nM) (N+B Inh) for 24 hours. The total cell numbers and cell viability between empty vector (EV) vs. CKS1B-cDNA transfected OCI-MY5 cell line are 4.85M vs. 5.11M and 92% vs. 90% in DMSO, 3.10M vs. 1.61M and 61% vs. 35% in Nif, 3.46M vs. 2.10M and 78% vs. 53% in U0126, 2.87M vs. 0.85M and 64% vs. 32% in BCL2 Inh, 0.90M vs. 0.17M and 39% vs. 14% in N + U, and 0.36M vs. 0.07M and 29% vs. 11% in N + B, respectively. (C) Cell growth and (D) cell death were evaluated in CKS1B-transfected XG1 treated with N+U Inh and N+B Inh for 24 hours. The total cell numbers and cell viability between empty vector (EV) vs. CKS1B-cDNA transfected XG1 cell line are 4.12M vs. 4.30M and 90% vs. 92% in DMSO, 2.17M vs. 1.64M and 62% vs. 35% in Nif, 2.63M vs. 1.83M and 78% vs. 52% in U0126, 2.03M vs. 0.96M and 65% vs. 32% in BCL2 Inh, 0.97M vs. 0.20M and 33% vs. 2% in N + U, and 0.58M vs. 0.09M and 27% vs. 3% in N + B, respectively. The Combination Indices values of EV-and CKS1B -transfected OCI-MY5 and XG-1 cells treated with combination of STAT3 and MEK1 inhibitors are 0.44 and 0.30, and 0.69 and 0.27, respectively; while these cells treated with combination of STAT3 and BCL2 inhibitors are 0.37 and 0.27, and 0.50 and 0.26, respectively. Both combinations showed synergistic effects in inhibition MM cell growth and induction MM cell apoptosis. Untreated cells and EV-transfected cells with or without treatment served as controls. Results were expressed as Mean ± SD of three independent experiments ( *p < 0.05 and **p < 0.01).