Journal: Journal of Bacteriology

Article Title: Arginine-Rich Small Proteins with a Domain of Unknown Function, DUF1127, Play a Role in Phosphate and Carbon Metabolism of Agrobacterium tumefaciens

doi: 10.1128/JB.00309-20

Figure Lengend Snippet: Seven DUF1127 proteins are predicted and produced in A. tumefaciens. (A) Characteristics of three SDPs and four LDPs. *, protein annotation was incorrect and adjusted at the N terminus (see Fig. S2B in the supplemental material). (B) Western blot analysis of chromosomally integrated C-terminal 3×FLAG fusions of all seven DUF1127 proteins at different growth phases in YEB medium at 30°C. A heat shock was applied for 20 min at 42°C and cold shock for 20 min at 17°C.

Article Snippet: The stacked bar chart shows the absolute frequency for the lengths of all DUF1127 proteins from InterPro.

Techniques: Produced, Western Blot

Journal: Journal of Bacteriology

Article Title: Arginine-Rich Small Proteins with a Domain of Unknown Function, DUF1127, Play a Role in Phosphate and Carbon Metabolism of Agrobacterium tumefaciens

doi: 10.1128/JB.00309-20

Figure Lengend Snippet: DUF1127 proteins can be divided into clusters. (A) SDPs from A. tumefaciens show a high sequence similarity. Identical residues are marked with an asterisk, residues with highly similar properties are marked with a colon, and weakly conserved residues are marked with a period. Arginine residues are shaded in black. (B) The homology tree of all seven DUF1127 proteins from A. tumefaciens and YjiS from E. coli was calculated by Clustal Omega (109). (C) Subdivision of DUF1127 proteins. The stacked bar chart shows the absolute frequency for the lengths of all DUF1127 proteins from InterPro. A kernel density estimation (dashed line) was superimposed. DUF1127 proteins were divided into subclasses according to sequence similarity to either SDPs from A. tumefaciens or to YjiS from E. coli.

Article Snippet: The stacked bar chart shows the absolute frequency for the lengths of all DUF1127 proteins from InterPro.

Techniques: Sequencing

Journal: Journal of Bacteriology

Article Title: Arginine-Rich Small Proteins with a Domain of Unknown Function, DUF1127, Play a Role in Phosphate and Carbon Metabolism of Agrobacterium tumefaciens

doi: 10.1128/JB.00309-20

Figure Lengend Snippet: Cooccurrence of DUF1127 proteins with LsrB and cuckoo sRNAs. The tree shows the phylogenetic relations of a selection of alpha- (α-proteo.) and gammaproteobacteria (γ-proteo.). For each strain, the numbers of SDPs, YjiS-like proteins, and LDPs are given. BLAST searches identified the proteins with the highest similarity to LsrB from A. tumefaciens (A9CI74, B5ZXG9, A0A068SS95, A0A081MRV5, C3MD12, W8I232, G7D4X2, Q2YRP4, Q3J274, Q9I1F9, A1JKA5, A0A0H2UWZ6, A0A0H3GNP2, P30864, and Q8ZRM6). Red and blue colors represent proteins with higher and lower sequence identity than 40%, respectively A genomic neighborhood to a trxB homolog is indicated by a check mark. Putative cuckoo sRNA sequences were extracted from each genome by searching for the pattern (CCTCCTCCC-N20–50)≥1-CCTCCTCCC. The numbers of sequences encoding two, three, or four CCUCCUCCC motifs are given.

Article Snippet: The stacked bar chart shows the absolute frequency for the lengths of all DUF1127 proteins from InterPro.

Techniques: Selection, Sequencing

Journal: Journal of Bacteriology

Article Title: Arginine-Rich Small Proteins with a Domain of Unknown Function, DUF1127, Play a Role in Phosphate and Carbon Metabolism of Agrobacterium tumefaciens

doi: 10.1128/JB.00309-20

Figure Lengend Snippet: SDP and LDP genes are reciprocally regulated by the transcription factor LsrB. (A) Northern blot analyses of SDP- and LDP-encoding genes in the WT and lsrB mutant. Ethidium bromide (EtBr)-stained 16S or 23S rRNA served as a loading control. (B) Putative LsrB-binding sites marked in red match the TGC-N7-GCA motif, and orange sequences display the shorter TGC-N6-GCA motif. Previously described binding sites of the three DUF1127 genes from B. abortus (bab1_0914, bab2_0514, and bab2_0574) are underlined. The position relative to the transcriptional start site (+1) is indicated.

Article Snippet: The stacked bar chart shows the absolute frequency for the lengths of all DUF1127 proteins from InterPro.

Techniques: Northern Blot, Mutagenesis, Staining, Binding Assay

Journal: Scientific Reports

Article Title: Faecal pollution affects abundance and diversity of aquatic microbial community in anthropo-zoogenically influenced lotic ecosystems

doi: 10.1038/s41598-019-56058-x

Figure Lengend Snippet: Relative abundance of bacterial populations among all studied sites (AG – Aas, Grytelandsbekken; SB – Ski, Blåveisbekken; BJ – Bergen, Jordalsvatnet; OM – Oslo, Maridalsvannet; and TJ – Trondheim, Jonsvannet): ( a ) all bacterial phyla, ( b ) Proteobacteria class distribution. The stacked bar charts were generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Article Snippet: The stacked bar charts were generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Techniques: Generated

Journal: Scientific Reports

Article Title: Faecal pollution affects abundance and diversity of aquatic microbial community in anthropo-zoogenically influenced lotic ecosystems

doi: 10.1038/s41598-019-56058-x

Figure Lengend Snippet: Relative abundance of bacterial populations among all studied sites (AG – Aas, Grytelandsbekken; SB – Ski, Blåveisbekken; BJ – Bergen, Jordalsvatnet; OM – Oslo, Maridalsvannet; and TJ – Trondheim, Jonsvannet): ( a ) bacterial phyla other than Proteobacteria and Bacteroidetes ; ( b ) genera distribution of the other bacterial phyla. The stacked bar charts were generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Article Snippet: The stacked bar charts were generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Techniques: Generated

Journal: Scientific Reports

Article Title: Faecal pollution affects abundance and diversity of aquatic microbial community in anthropo-zoogenically influenced lotic ecosystems

doi: 10.1038/s41598-019-56058-x

Figure Lengend Snippet: Relative abundance of archaeal populations among all studied sites (AG – Aas, Grytelandsbekken; SB – Ski, Blåveisbekken; BJ – Bergen, Jordalsvatnet; OM – Oslo, Maridalsvannet; and TJ – Trondheim, Jonsvannet): ( a ) phylum and class level; ( b ) genus level. The stacked bar charts were generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Article Snippet: The stacked bar charts were generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Techniques: Generated

Journal: Scientific Reports

Article Title: Faecal pollution affects abundance and diversity of aquatic microbial community in anthropo-zoogenically influenced lotic ecosystems

doi: 10.1038/s41598-019-56058-x

Figure Lengend Snippet: Phylogenetic alpha diversity of all studied sites (AG – Aas, Grytelandsbekken; SB – Ski, Blåveisbekken; BJ – Bergen, Jordalsvatnet; OM – Oslo, Maridalsvannet; and TJ – Trondheim, Jonsvannet). The alpha rarefaction plot was generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Article Snippet: The stacked bar charts were generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Techniques: Generated

Journal: Scientific Reports

Article Title: Faecal pollution affects abundance and diversity of aquatic microbial community in anthropo-zoogenically influenced lotic ecosystems

doi: 10.1038/s41598-019-56058-x

Figure Lengend Snippet: Beta diversity heat map with hierarchical clustering of the study sites (AG – Aas, Grytelandsbekken; SB – Ski, Blåveisbekken; BJ – Bergen, Jordalsvatnet; OM – Oslo, Maridalsvannet; and TJ – Trondheim, Jonsvannet). The hierarchical clustering heat map was generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Article Snippet: The stacked bar charts were generated using the tools in CLC Microbial Genomics Module version 2.5.1 (CLC Bio, QIAGEN Company, Aarhus, Denmark, https://www.qiagenbioinformatics.com/clc-microbial-genomics-module-latest-improvements ).

Techniques: Generated

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet: Knockout of NEK2 improves Eμ- myc mouse survival and induces T cell activation (A) Overall survival of Eμ- myc mice with indicated Nek2 genotypes. (B–D) Numbers of circulating lymphocytes and platelets in peripheral blood (B); spleen (SPL) and thymus weight index as calculated by normalizing to body weight (C); flow cytometry analyses of percentages of pre- and pro-B cells (B220 low IgM − ), immature B cells (B220 low IgM + ), and mature B cells (B220 high IgM + ) from bone marrow (BM) and SPL (D) in indicated groups. n = 9–10 mice/group from three independent experiments. Boxplot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. (E–G) Volcano plot showing differential gene expression, where red dots represent genes with a |log2FC| > 2 and p value <0.01 (E); Gene Ontology (GO) analysis of upregulated genes (F); and heatmap showing gene expression level of selected chromosomal instability genes and IFN-γ signature genes (G) in CD19 + SPL cells of Eμ- myc / Nek2 −/− vs. Eμ- myc / Nek2 +/+ mice. n = 2 mice/group. (H) Western blot analysis of cell lysates to detect JAK/STAT1/IRF1/PD-L1 pathway in CD19 + SPL cells of Eμ- myc / Nek2 −/− vs. Eμ- myc / Nek2 +/+ mice. (I) Flow cytometry analyses showing percentages of T naive cells (T N , CD62L + CD44 − ), T central memory (T CM , CD62L + CD44 + ), and T effector memory (T EM , CD62L − CD44 + ) of CD8 + T cells from BM and SPL in indicated groups. n = 9–10 mice/group from three independent experiments. Boxplot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Please also see Figure S1 and Table S1 .

Article Snippet: ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined.

Techniques: Knock-Out, Activation Assay, Flow Cytometry, Comparison, Expressing, Western Blot

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet: Loss of NEK2 reduces tumor-associated macrophages (TAMs) and Tregs (A) Experimental layout. (B) Serum IgG2b levels. n = 6–8 mice/group from two independent experiments with three technical replicates. Boxplot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. ∗∗∗p < 0.001. (C) Survival analysis. n = 10 mice/group. ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined. Boxplot shows mean ± SD and the p value for two-tailed t test. ∗p < 0.05; ∗∗∗p < 0.001. (J) KEGG analysis of upregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (K) UMAP plots of macrophage subpopulations (left) and violin plots showing their marker genes (right). (L) UMAP plots of NK/T subpopulations (left) and violin plots showing their marker genes (right). Please also see Figure S2 and Table S2 .

Article Snippet: ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined.

Techniques: Comparison, Expressing, Staining, Two Tailed Test, Marker

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet: NEK2 deficiency decreased the immunosuppressive activity of TAMs and MDSCs (A and B) Flow cytometric analysis showing the percentages of indicated cell populations in the BM and SPL of Nek2 +/+ , Nek2 −/− , 5TGM1/ Nek2 +/+ , and 5TGM1/ Nek2 −/− mice. n = 4–5 mice/group from two independent experiments. Dot plot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. (C) Experimental layout. BM-derived macrophages were generated from Nek2 −/− and Nek2 +/+ C57BL/KaLwRij mice after 7 days of M-CSF treatment. TAMs were induced by adding 5TGM1 conditioned medium (CM) for an additional 2 days followed by detection of macrophage survival, cell cycle, CD206 expression, and its effects on T cells proliferation and IFN-γ secretion. (D) Prestoblue staining to detect mouse macrophage survival. n = 3 independent experiments, each with three technical replicates. Boxplot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. (E) Cell-cycle analyses of mouse macrophages. n = 3 independent experiments, each with three technical replicates. Boxplot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. (F) Flow cytometry analyses of CD206 expression on mouse macrophages. n = 3 independent experiments, each with three technical replicates. (G) Representative histograms and quantification analysis showing the percentages of divided CD8 + T cells co-cultured with TAMs on day 3 (left); IFN-γ levels in the co-culture supernatants on day 1 (right). n = 3 independent experiments, each with three technical replicates. Boxplot shows mean ± SD and the p value for two-tailed t test. (H) Violin plot showing Mif and Hmgb1 gene expression in MM and plasma cell populations identified in Figure 2 D. (I) Western blots analysis of cell lysates to detect PI3K and STAT3 pathway proteins in mouse TAMs and naive macrophages. (J) Representative histograms and quantification analysis showing the percentages of divided CD8 + T cells co-cultured with MDSCs on day 0 and day 3 (left); IFN-γ levels in the co-culture supernatants on day 1 (right). n = 3 independent experiments, each with three technical replicates. Boxplot shows mean ± SD and the p value for two-tailed t test. (K and L) Western blots of cell lysates to detect C/EBPβ, arginase-1 (K), as well as PI3K and STAT3 pathway proteins (L) of MDSCs and freshly isolated CD11b + cells. ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Please also see Figure S3 and Table S3 .

Article Snippet: ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined.

Techniques: Activity Assay, Comparison, Derivative Assay, Generated, Expressing, Staining, Flow Cytometry, Cell Culture, Co-Culture Assay, Two Tailed Test, Western Blot, Isolation

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet: MM patients with high expression of NEK2 show an impaired T cell response (A) NEK2 mRNA levels in UAMS-MM dataset among MGUS, SMM, NDMM, and RRMM (left), seven MM subtypes of NDMM (middle), and high risk (HR) vs. low risk (LR) (right). The seven MM subtypes include proliferation (PR), low bone disease (LB), MMSET (MS), hyperdiploid (HY), CCND1 (CD-1), CCND3 (CD-2), and MAF/MAFB (MF). NEK2 expression was identified using probe 204641_at. Violin plot shows median ± 95% confidence interval and p value for one-way ANOVA followed by a Tukey’s multiple comparison test. (B) NEK2 mRNA levels from 606 NDMM samples of MMRF CoMMpass dataset among seven MM subtypes. NEK2 expression was identified with ENSG00000117650. Violin plot shows median ± 95% confidence interval and p value for one-way ANOVA followed by a Tukey’s multiple comparison test. (C) Kaplan-Meier analyses of overall survival (left) and progression-free survival (right) of NDMM patients from the MMRF CoMMpass dataset based on NEK2 expression. Median OS of NEK2 high : 2,207 days, NEK2 Low : undefined. Median PFS of NEK2 high : 804 days, NEK2 Low : 1,260 days. (D) Mass cytometry analyses of T cell populations from primary human MM bone marrow mononuclear cells (BM-MNCs). n = 6/group. Dot plot shows mean ± SD and the p value for the two-way ANOVA followed by Sidak multiple comparison test. (E) Experimental layout. Freshly isolated BM-MNCs of MM patients were processed to magnetic cell sorter to obtain paired CD138 + MM cells and CD138 – cells. CD138 + MM cells were processed for RNA extraction to detect NEK2 expression by qPCR. CD138 – cells were enriched for CD8 + T cells and cultured with Dynabeads Human T-Activator CD3/CD28 for T cell proliferation detection and cytokine detection of the culture supernatant. (F) Flow cytometry analyses showing PD1 expression in CD8 + and CD4 + T cells from MM BM-MNCs (n = 14) and their correlation with NEK2 mRNA expression levels of MM cells. Red line represents simple linear regression. Pearson correlation coefficient (r) was noted in the figure. (G) Proliferation analyses of human CD8 + T cells (n = 14) after stimulation with Dynabeads Human T-Activator CD3/CD28 for 5 days (left). Correlation analysis of percentages of undivided CD8 + T cells with NEK2 mRNA expression levels of MM cells (right). (H) Correlation analyses of NEK2 mRNA expression levels of MM cells with IFN-γ, IL6, and TNFα concentrations in the culture supernatant of CD8 + cells after stimulation with Dynabeads Human T-Activator CD3/CD28 for 24 h (n = 16). ∗p < 0.05; ∗∗∗p < 0.0001. Please also see Figure S4 .

Article Snippet: ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined.

Techniques: Expressing, Comparison, Mass Cytometry, Isolation, RNA Extraction, Cell Culture, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet: NEK2 suppresses the IFN-γ/PD-L1 signaling pathway in MM patients (A) Heatmap of CIN and IFN-γ signaling genes from NDMM patients in the TT2 cohort (n = 351). (B) Correlation analyses of CD274 and NEK2 mRNA from NDMM samples derived from MMRF CoMMpass (n = 606) and UAMS-MM (n = 1,497) datasets. CD274 expression was identified using probe 223834_at in UAMS-MM and ENSG00000120217 in the MMRF CoMMpass dataset. Red line represents simple linear regression. Pearson correlation coefficient (r) was noted in the figure. (C) Dot plots of CD274 mRNA expression among seven MM subtypes of NDMM samples from MMRF CoMMpass (n = 606) and UAMS-MM (n = 1,388 that are subtypes available) datasets. Violin plot shows median ± 95% confidence interval and p value for one-way ANOVA followed by a Tukey’s multiple comparison test. (D) Kaplan-Meier analyses of overall survival (left) and progression-free survival (right) from NDMM patients of MMRF CoMMpass and UAMS-MM datasets based on NEK2 and CD274 expression. (E) IHC staining of NEK2 and PD-L1 expression on 50 bone marrow biopsies derived from NDMM patients (40×, scale bar represents 60 μm). Correlation analysis of percentages of NEK2-positive nuclei and PD-L1-positive cells. Red line represents simple linear regression. Spearman correlation coefficient (r) was noted in the figure. ∗∗∗p < 0.0001.

Article Snippet: ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined.

Techniques: Derivative Assay, Expressing, Comparison, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet: NEK2 inhibition increases potency of anti-PD-L1 monoclonal antibody (mAb) against MM (A) Experimental layout for a combination treatment of INH154 and PD-L1 mAb in freshly isolated BM-MNCs in vitro . Survived CD138 + MM cells, CD14 + myeloid cells, CD8 + T cells, and CD4 + T cells and their PD-L1 or PD1 expression were detected 24 h after the treatment by flow cytometry. (B) Flow cytometry analysis showing percentages of CD138 + MM cells and CD14 + monocytes of total live cells after treatments. (C and D) Flow cytometry analysis showing PD-L1 expression in CD138 + MM cells (C) and CD14 + monocytes (D) after treatments. (E) Flow cytometry analysis showing percentages of CD3 + of total live cells and CD8 + and CD4 + T cells of total CD3 + T cells after treatments. (F) Flow cytometry analysis showing PD1 expression in CD8 + T cells and CD4 + T cells after treatments. (B–F) Spaghetti plots show data from each patient and p value for randomized block (repeated measures) ANOVA followed by a Tukey’s multiple comparison test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Article Snippet: ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined.

Techniques: Inhibition, Isolation, In Vitro, Expressing, Flow Cytometry, Blocking Assay, Comparison

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet: Combining NEK2 inhibition with anti-PD-L1 mAb therapy provides therapeutic benefits in MM mouse model (A) Experimental timeline for a combination treatment of INH154 and PD-L1 mAb in C57BL/6 mice injected with Vκ12653 MM cells. MM was monitored by serum protein electrophoresis to detect γ-globulin. Survival was monitored up to 80 days post Vκ12653 MM cells injection. Endpoint of MM engraftment was analyzed BM and SPL by fluorescence-activated cell sorting on day 35 post Vκ12653 MM cell injection when mice in the control group were moribund. (B) Quantitative analyses of mouse serum γ-globulin percentages on day 35 post Vκ12653 MM cell injection. n = 5 mice/group with two biological replicates. Boxplot shows mean ± SD and p value for one-way ANOVA followed by a Tukey’s multiple comparison test. (C) Survival analysis. (D) SPL weight index. n = 5 mice/group with two biological replicates. Boxplot shows mean ± SD and p value for one-way ANOVA followed by a Tukey’s multiple comparison test. (E and F) Flow cytometry analysis showing percentages of CD138 + B220 – MM cells and PD-L1 expression in MM cells in BM (E) and SPL (F). n = 5 mice/group with two biological replicates. Boxplot shows mean ± SD and p value for one-way ANOVA followed by a Tukey’s multiple comparison test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Please also see Figure S5 .

Article Snippet: ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined.

Techniques: Inhibition, Injection, Protein Electrophoresis, Fluorescence, FACS, Comparison, Flow Cytometry, Expressing

Journal: Cell Reports Medicine

Article Title: High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma

doi: 10.1016/j.xcrm.2023.101214

Figure Lengend Snippet:

Article Snippet: ∗∗p < 0.01 (D) Stacked bar charts representing of BM cell populations following 10X Genomics scRNA-seq workflow (n = 6,379 cells for Nek2 +/+ , 5,746 cells for Nek2 −/− , 8,483 cells for 5TGM1/ Nek2 +/+ , and 9,676 cells for 5TGM1/ Nek2 −/− group). (E) Violin plot representing NEK2 expression in immune cell populations identified in (D). (F) Volcano plot showing differential gene expression in the macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group with a |log 2 FC| > 1 and p < 0.05. (G) KEGG analysis of downregulated genes in macrophages of 5TGM1/ Nek2 −/− vs. 5TGM1/ Nek2 +/+ group. (H) Representative reconstructed μCT images of tibia sagittal sections showing bone lytic lesions and trabecular architecture. n = 5–10 mice/group from two independent experiments. (I) Quantitative histomorphometric analyses of TRAP-stained number of osteoclast surface per bone surface (Oc.S/BS) and osteoclast per bone perimeter (N.Oc/B.Pm). n = 10 mice/group from two independent experiments. n = 3 TRAP staining area from each slide were randomly selected and examined.

Techniques: Purification, Recombinant, Western Blot, Stripping, Cell Stimulation, Staining, Cell Isolation, Activation Assay, Extraction, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, SYBR Green Assay, RNA Sequencing Assay, Mutagenesis, Knock-Out, Software