Fig 1: Cdc37 is gradually upregulated during plasma cell maturation and positively correlates with Xbp1s.a Bone marrow MM cells were enriched by sequential FACS of lymphoid FSC/SSC, negative selection for CD2 (T and NK cells) and CD14 (monocytes), as well as gated into a subpopulation by CD38 and CD138 status. b Sorted CD38−CD138− pre-plasmablast, CD38+CD138− plasmablast, and CD38+CD138+ plasma cells were subjected to qRT-PCR analysis for gene expression of Cdc37 and Xbp1s. c CD38−CD138− pre-plasmablast, CD38+CD138− plasmablast, and CD38+CD138+ plasma cells were subjected to immunofluorescence analysis for protein expression of Cdc37 and Xbp1s. d Immunofluorescence analysis of Cdc37 and Xbp1s in CD138+ cells derived from a newly diagnosed patient responding to BTZ-based treatment (MM patient 1) and a patient displaying resistance to BTZ (MM patient 2). e Correlation analysis of gene expression of Cdc37 and Xbp1s from 45 newly diagnosed and 18 relapsed MM patients from qRT-PCR data. f Data obtained from GSE2658 showing prognostic relevance of Xbp1s mRNA on MM. Kaplan–Meier analyses revealed that MM patients with low expression of Xbp1s had inferior event-free survival in newly diagnosed TT2 and TT3 cohorts.
Fig 2: Suppression of Cdc37 induces immaturity of plasma cells.a First panel: The cell culture supernatant of NCI-H929 Scramble and Cdc37shRNA cells was subjected to ELISA analysis for the concentrations of human immunoglobulin light-chain proteins (*p < 0.05). Second panel: Total RNA of NCI-H929 Scramble and Cdc37shRNA cells was isolated and subjected to qRT-PCR analysis for Cdc37 and Xbp1s (**p < 0.01). Third panel: Whole-cell lysates of NCI-H929 Scramble and Cdc37shRNA cells were subjected to western blot analysis for Cdc37 and Xbp1s. Fourth panel: Flow cytometric analysis of surface CD38 and CD49e on NCI-H929 Scramble and Cdc37shRNA cells. b First panel: The cell culture supernatant of KMS11 Scramble and Cdc37shRNA cells was subjected to ELISA analysis for the concentrations of human immunoglobulin light-chain proteins (**p < 0.01). Second panel: Total RNA of KMS11 Scramble and Cdc37shRNA cells was isolated and subjected to qRT-PCR analysis for Cdc37 and Xbp1s (*p < 0.05, **p < 0.01). Third panel: Whole-cell lysates of KMS11 Scramble and Cdc37shRNA cells were subjected to western blot analysis for Cdc37 and Xbp1s. Fourth panel: Flow cytometric analysis of surface CD38 and CD49e on KMS11 Scramble and Cdc37shRNA cells. c NCI-H929 cells were treated with 100 and 200 nM celastrol for 24 and 48 h, respectively. Left panel: The cell culture supernatant was subjected to ELISA for the concentration of human Ig light-chain proteins (*p < 0.05, **p < 0.01, ***p < 0.001). Middle panel: The total RNA was isolated and subjected to qRT-PCR analysis for Cdc37 and Xbp1s (*p < 0.05, **p < 0.01). Right panel: The whole-cell lysates were subjected to western blot analysis for Xbp1s, CD49e, and Cdc37.
Fig 3: Low expression of Cdc37 is linked to BTZ resistance in multiple myeloma.a Heatmap showing Cdc37 mRNA expression in a sequential MM sample set [at diagnosis (Baseline), pre first transplantation (Pre T1), pre second transplantation (Pre T2), and post second transplantation (Post T2)], data obtained from GSE19554. b Cdc37 mRNA expression was detected in CD138+ cells from 60 newly diagnosed MM patients (ND) and 25 relapsed cases (Relapsed). The relative Cdc37 mRNA expression in ND and relapsed MM patients was 0.78 ± 1.13 and 0.25 ± 1.07, respectively (*p < 0.05). c Cdc37 protein expression in primary MM cells was examined with ICC. Left panel: MM cells derived from 14 newly diagnosed patients (ND) and 10 relapsed patients (Relapsed) were subjected to ICC for Cdc37 protein expression, and representative cases are shown. Right panel: The semiquantitative scoring is shown. The relative Cdc37 protein expression in ND and relapsed MM patients was 1.90 ± 0.85 and 0.81 ± 0.44, respectively (**p < 0.01). d Cdc37 mRNA expression (upper) and protein level (lower) were examined in ANBL6.WT/ANBL6.BR cells (left), RPMI-8226/RPMI-8226.Dox40 cells (middle), and MM.1S/MM.1R cells (right) by qRT-PCR and western blot. e Cdc37 mRNA expression was detected in CD138+ cells from 14 patients relapsed after BTZ-based treatment, while 11 cases relapsed after other therapy without BTZ. The relative Cdc37 mRNA expression was -0.21 ± 1.05 and 0.83 ± 0.83, respectively (*p < 0.05).
Fig 4: Suppression of Cdc37 induces BTZ resistance in MM cells.a NCI-H929 cells were infected with scramble (NCI-H929 Scramble) and Cdc37shRNA (NCI-H929 Cdc37shRNA) lentivirus. Left panel: The apoptosis rate was detected by flow cytometry. Middle panel: The statistical analysis of apoptosis cell distribution from three repeated experiments (*p < 0.05, **p < 0.01). Right panel: Caspase-3, CL-Caspase-3, and GAPDH were detected by western blot analysis. b KMS11 cells were infected with scramble (KMS11 Scramble) and Cdc37shRNA (KMS11 Cdc37shRNA) lentivirus. Left panel: The apoptosis rate was detected by flow cytometry. Middle panel: The statistical analysis of apoptosis cell distribution from three repeated experiments (*p < 0.05, **p < 0.01). Right panel: Caspase-3, CL-Caspase-3, and GAPDH were detected by western blot analysis. c NCI-H929 cells were treated with celastrol (Cel), BTZ, or their combination for 24 h. Left panel: The apoptosis analysis with flow cytometry. Middle panel: The statistical analysis of apoptosis cell distribution was performed from three repeated experiments (*p < 0.05, **p < 0.01). Right panel: Caspase-3, CL-Caspase-3, and GAPDH were detected by western blot analysis.
Fig 5: Disruption of Cdc37 leads to BTZ resistance in vivo.a The schematic diagram of the 5T33MMvt-KaLwRij model. b Kaplan–Meier analyses of overall survival of different experimental groups. c The concentrations of Ig light-chain proteins in the serum of different groups were determined with ELISA (*p < 0.05, **p < 0.01, ***p < 0.001). d B220+CD138+IgM+ plasmablast cells from mice bone marrow were selected with flow cytometry, and the percentages of plasmablast cells were calculated in the respective groups (*p < 0.05, **p < 0.01, ***p < 0.001). e Extramedullary myeloma tissues removed from 5T33MMvt-KaLwRij mice were subjected to immunostaining for CD138 (green) and Xbp1s (orange). f The model of our hypothesis in this work.
Supplier Page from Abcam for Anti-Cdc37 antibody [EPR4934]