Journal: Cell Death Discovery

Article Title: SENP1 promotes MCL pathogenesis through regulating JAK-STAT5 pathway and SOCS2 expression

doi: 10.1038/s41420-021-00578-x

Figure Lengend Snippet: A Representative immunohistochemical images of SENP1 staining in human MCL samples and control samples ( n = 4 per group). B The mean integrated optical density (IOD) of SENP1 protein in human MCL samples and control samples was calculated using Image-Pro Plus 6.0 software. C SENP1 mRNA level in human MCL samples and control samples was analyzed through extracting GEO RNA-seq data. SENP1 protein level was determined by western blot ( D ), SENP1 mRNA level was measured by RT-qPCR ( E ) in human normal B cell line (Wil2-s) and MCL cell lines (MAVER-1, REC-1, Mino and Jeko-1) . F Nuclear and cytoplasmic protein level of SENP1 was determined by western blot after subcellular fractionation in Jeko-1 and Mino cells. Data were presented as mean ± SD and analyzed by two-sided Student’s t test. n = 3; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Furthermore, we detected the protein and mRNA expressions of SENP1 in several human MCL cell lines (MAVER-1, REC-1, Mino, Jeko-1) and human normal B cell line Wil2-s, which demonstrated that SENP1 mRNA and protein expressions were upregulated in REC-1, Mino, and Jeko-1 cells compared with Wil2-s cells (Fig. ).

Techniques: Immunohistochemical staining, Staining, Control, Software, RNA Sequencing, Western Blot, Quantitative RT-PCR, Fractionation

Journal: Cell Death Discovery

Article Title: SENP1 promotes MCL pathogenesis through regulating JAK-STAT5 pathway and SOCS2 expression

doi: 10.1038/s41420-021-00578-x

Figure Lengend Snippet: A Heatmap revealed differentially expressed genes between Jeko-1 cells transfected with shCon and shSENP1-3. B KEGG enrichment analysis showed that differentially expressed genes were enriched at different signaling pathways after SENP1 knockdown. C GSEA was performed using differentially expressed genes. D SOCS2 mRNA level in human MCL samples and normal control samples was analyzed through extracting GEO RNA-seq data. E Pearson correlation analysis between SENP1 mRNA level and SOCS2 mRNA level using GEO RNA-seq data. SOCS2 mRNA level was measured by RT-qPCR ( F ), SOCS2 ( G ) and p-STAT5 ( H ) protein levels were measured by western blot in transfected Jeko-1 and Mino cells. Data were presented as mean ± SD and analyzed by two-sided Student’s t test. n = 3; * P < 0.05, and *** P < 0.001.

Article Snippet: Furthermore, we detected the protein and mRNA expressions of SENP1 in several human MCL cell lines (MAVER-1, REC-1, Mino, Jeko-1) and human normal B cell line Wil2-s, which demonstrated that SENP1 mRNA and protein expressions were upregulated in REC-1, Mino, and Jeko-1 cells compared with Wil2-s cells (Fig. ).

Techniques: Transfection, Protein-Protein interactions, Knockdown, Control, RNA Sequencing, Quantitative RT-PCR, Western Blot

Journal: Cell Death Discovery

Article Title: SENP1 promotes MCL pathogenesis through regulating JAK-STAT5 pathway and SOCS2 expression

doi: 10.1038/s41420-021-00578-x

Figure Lengend Snippet: A Images of the MCL xenografts in each mouse subcutaneously injected with shCon or shSENP1-3 transfected Jeko-1 cells ( n = 5 per group). B Tumor volume in shCon and shSENP1-3 groups was recorded every two days for total 17 days. C Tumor growth rate was compared between shCon and shSENP1-3 groups. D Tumor weight was compared between shCon and shSENP1 groups. E Representative immunohistochemical images of SENP1 staining in MCL xenografts from shCon and shSENP1-3 groups. F The mean integrated optical density (IOD) of SENP1 protein in MCL xenografts from shCon and shSENP1-3 groups was calculated by Image-Pro Plus 6.0 software. G SENP1 protein level in each MCL xenograft from shCon and shSENP1-3 groups was measured by western blot. H Representative images of Ki67 staining, TUNEL assay and p-STAT5 in MCL xenografts from shCon and shSENP1-3 groups. I The mean integrated optical density (IOD) of Ki67, TUNEL and p-STAT5 in MCL xenografts from shCon and shSENP1-3 groups were calculated by Image-Pro Plus 6.0 software. Data were presented as mean ± SD and analyzed by two-sided Student’s t test. n = 3; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Furthermore, we detected the protein and mRNA expressions of SENP1 in several human MCL cell lines (MAVER-1, REC-1, Mino, Jeko-1) and human normal B cell line Wil2-s, which demonstrated that SENP1 mRNA and protein expressions were upregulated in REC-1, Mino, and Jeko-1 cells compared with Wil2-s cells (Fig. ).

Techniques: Injection, Transfection, Immunohistochemical staining, Staining, Software, Western Blot, TUNEL Assay

Journal: Cell Reports Medicine

Article Title: Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma

doi: 10.1016/j.xcrm.2024.101484

Figure Lengend Snippet: DNMT3A expression is required for in vitro and in vivo growth of mantle cell lymphoma (A) The design of 2 sgRNAs targeting DNMT3A (top) and immunoblots (bottom) showing the reduction of DNMT3A expression in 7 distinct clones of MCL cell lines after 6 days of doxycycline treatment (20 ng/mL). (B) The global methylation was reduced after DNMT3A KO using methylation dot blots. The global methylation was reduced after treating Granta-519 cells with decitabine (1 μM, 48 h), and this served as a positive control for this assay (top). A representative image of reduced global methylation in Rec-1- sgDNMT3A clone no. 6 (Rec-1 no. 6) after doxycycline-induced DNMT3A KO 6 days posttreatment (bottom). (C) DNMT3A KO inhibited the in vitro growth of MCL cell lines. Error bars represent mean ± SD (Student’s t test). (D) Z-138 no. 7 (left) and Granta-519 no. 7 (right) were established as subcutaneous tumors in NSG mice. Error bars represent mean ± SEM (2-way ANOVA). All in vitro experiments were performed independently at least 3 times with 3–4 technical replicates.

Article Snippet: Doxycycline-inducible human MCL cell lines (Mino, Rec-1, JeKo-1, Z138), originally obtained from the American Type Culture Collection (ATCC), were engineered and authenticated by gene expression profiling to express the bacterial tetracycline repressor as described previously.

Techniques: Expressing, In Vitro, In Vivo, Western Blot, Clone Assay, Methylation, Positive Control

Journal: Cell Reports Medicine

Article Title: Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma

doi: 10.1016/j.xcrm.2024.101484

Figure Lengend Snippet: DNMT3A expression is required for OXPHOS in MCL cells by controlling mitochondrial biogenesis independent of its methyltransferase activity (A) GSEA of DNMT3A KO cells shows downregulation of genes involved in OXPHOS, mitochondrial biogenesis, and MYC transcriptional pathway compared to control cells. (B) The reduced rate of OCR (pmol/min/cell) and ATP productions in DNMT3A KO cells relative to control cells. All of the OCR values were normalized to total live-cell number. OCR experiments were performed independently at least 2 times, each with 6 technical replicates. (C) Reduced MMP in DNMT3A KO cells compared to control cells by flow cytometric analysis of JC-1 dye. (D) Representative images of TEM of Z-138 no. 7 control cells compared to DNMT3A KO cells (top). A significant decrease in mitochondrial mass, approximated by the relative area of mitochondria to cytosol per cell, of DNMT3A KO cells relative to control cells (bottom). Each data point represents an individual cell (n = 60–61 cells). TEM experiments were performed independently twice, each with duplicates. (E) DNMT3A V716G expression was sufficient to increase MMP in Rec-1 and JeKo-1 cells and the quantification of MMP in Rec-1 cells. Error bars represent mean ± SD (Student’s t test, 2 independent experiments in triplicate).

Article Snippet: Doxycycline-inducible human MCL cell lines (Mino, Rec-1, JeKo-1, Z138), originally obtained from the American Type Culture Collection (ATCC), were engineered and authenticated by gene expression profiling to express the bacterial tetracycline repressor as described previously.

Techniques: Expressing, Activity Assay, Control

Journal: Cell Reports Medicine

Article Title: Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma

doi: 10.1016/j.xcrm.2024.101484

Figure Lengend Snippet: Decitabine inhibits OXPHOS and the growth of ibrutinib-resistant MCL cells (A) A schematic of ex vivo OMI experiments in Z-138 xenografts. (B) Representative images of the fluorescence intensity of NAD(P)H and FAD in PBS (control, n = 3) and decitabine-treated mice (n = 3). Scale bar, 25 μm. (C) Representative images of τ m of FAD in control and decitabine-treated mice (left) and the quantification of FAD τ m (right) at the single-cell level (control = 2,544 cells; decitabine = 1,743 cells). Bars represent mean ± SD. Different colors denote separate imaging days. Scale bar, 25μm. (D) Single-cell quantifications of the lifetime of bound FAD (τ 1 ) (ps, picosecond), the percentage of bound FAD (α 1 ), the lifetime of free FAD (τ 2 ), and the percentage of free FAD (α 2 ). (E) Representative of images of the optical redox ratio and the single-cell quantifications of optical redox ratio. Different colors denote separate imaging days. Scale bar, 25 μm. Mann-Whitney U test was used in (C)–(E). (F) Z138 xenografts, the same as in (A). Red arrows indicate treatment start and end dates. Tumor dimensions were measured with calipers 3 times per week until endpoint, defined as any dimension exceeding 20 mm or mice becoming morbid. (G) The effect of decitabine in MCL-7 PDXs. (H) Bioluminescence images of MCL-4 PDX mice (left), growth curves presented as log 10 of the average radiance of tumors (ps −1 cm −2 sr −1 ), with dashed line indicating that all of the mice are euthanized (center), and the probability of survival (right) are shown. Error bars represent mean ± SEM (2-way ANOVA). sr: steradian or square radian.

Article Snippet: Doxycycline-inducible human MCL cell lines (Mino, Rec-1, JeKo-1, Z138), originally obtained from the American Type Culture Collection (ATCC), were engineered and authenticated by gene expression profiling to express the bacterial tetracycline repressor as described previously.

Techniques: Ex Vivo, Fluorescence, Control, Imaging, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma

doi: 10.1016/j.xcrm.2024.101484

Figure Lengend Snippet: Decitabine synergizes with a novel complex I inhibitor to inhibit the growth of cell lines and primary MCL cells (A) Highest single agent synergy scores of decitabine and IM156, an inhibitor of mitochondrial complex I, in MCL cell lines. A 4 × 4 matrix of 2 drugs and all of the possible combinations were designed on a 96-well plate. Synergy scores were calculated using SynergyFinder + . (B) Decitabine and IM156 synergized to inhibit primary MCL cells. (C) Decitabine and IM156 did not synergistically kill normal B cells.ns, not significant. (D) MCL cells were pretreated with gamitrinib (1.25 μM) or solvent control for 4 days; fresh drug was added every 2 days. Drug/solvent control was removed on day 4. Cells were incubated in normal growth media for 24 h before treatment with decitabine, IM156, or the combination of both drugs at indicated concentrations in a 96-well plate. On day 3 of treatment, cell viability was measured using CellTiter Glo. Error bars represent mean ± SD (Student’s t test). All of the experiments were performed independently twice, except for primary normal B cells, each with 3 technical replicates.

Article Snippet: Doxycycline-inducible human MCL cell lines (Mino, Rec-1, JeKo-1, Z138), originally obtained from the American Type Culture Collection (ATCC), were engineered and authenticated by gene expression profiling to express the bacterial tetracycline repressor as described previously.

Techniques: Solvent, Control, Incubation

Journal: Cell Reports Medicine

Article Title: Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma

doi: 10.1016/j.xcrm.2024.101484

Figure Lengend Snippet: DNMT3A is a potential biomarker for and therapeutic vulnerability of ibrutinib resistance (A) Representative intracellular flow cytometric of DNMT3A. The staining was validated in DNMT3A KO cells (top left). MCL-5 was less ibrutinib resistant (top right). MCL-4, MCL-7, and MCL-9, defined as ibrutinib-resistant samples, showed an increased DNMT3A expression after ibrutinib treatment (2.5 μM, 3 days). (B) Increased DNMT3A expression was correlated with resistance to ibrutinib. (C) DNMT3A KO desensitized MCL cell lines to ibrutinib. (D) The IC 50 of ibrutinib in control and DNMT3A KO MCL clones (Student’s t test, ∗∗p < 0.01). (E) The overexpression of DNMT3A WT or DNMT3A V716G desensitized the ibrutinib-sensitive JeKo-1 cells to ibrutinib. The IC 50 of ibrutinib is shown. (F) Decitabine enhanced the efficacy of ibrutinib in MCL cell lines. Relative viability for each condition, control (uninduced) or DNMT3A KO, were obtained by normalizing the number of live cells in treated conditions to that in the DMSO-treated control. The statistical significance of individual drug concentrations when combined one another is shown. Error bars represent mean ± SD (Student’s t test). (G) The effect of decitabine/ibrutinib alone or in combination in MCL-7 PDX model. Bioluminescence images (left), the radiance of primary tumors (top right), and mouse body weight of mice before and during treatment as indicated between 2 arrows (bottom right) are shown. Error bars represent mean ± SEM (2-way ANOVA).

Article Snippet: Doxycycline-inducible human MCL cell lines (Mino, Rec-1, JeKo-1, Z138), originally obtained from the American Type Culture Collection (ATCC), were engineered and authenticated by gene expression profiling to express the bacterial tetracycline repressor as described previously.

Techniques: Biomarker Discovery, Staining, Expressing, Control, Clone Assay, Over Expression