Journal: Histochemistry and Cell Biology

Article Title: Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice

doi: 10.1007/s00418-020-01905-6

Figure Lengend Snippet: Distribution of TBC proteins in the organ of Corti at P1 and P7. a – e age P1; a TBCA expression is found in the inner area of Kölliker’s organ, the inner hair cell and a tectal cell. b TBCB is expressed in the cells of the Kölliker’s organ. c TBCC labelling is detected in the basal half of the two pillar cells. d Similar TBCD is expressed in the pillar cells. e TBCE staining is found in the basal cells of Kölliker’s organ and the basal half of the inner phalangeal cell. Diffuse staining is visible around the nucleus and below the apical surface of the inner hair cells. The three outer hair cells are also marked. f – j age P7: f TBCA is detected in the inner and outer pillar cells, phalangeal extensions of the three Deiters’ cells, tectal cells and Hensen’s cells. g Similar to TBCA, TBCB is detected in the inner and outer pillar cells, the three Deiters’ cells, the tectal cells and Hensen’s cells. h The expression of TBCC is found in the supporting cells (inner and outer pillar cells, the phalangeal extensions of the three Deiters’ cells, the tectal cells and Hensen’s cells). i TBCD staining is found in the basal parts of the inner pillar cell. j In addition to the inner pillar cells, TBCE is also detected in the and the basal half of the inner phalangeal cell phalangeal extensions of Deiters’ cells. ( TM tectorial membrane with unspecific staining) (Scale bar = 25 µm)

Article Snippet: For immunofluorescence analysis, sections were post-fixed in 4% PFA for 10 min, rinsed in 0.05 M phosphate-buffered saline three times and blocked in 10% bovine serum albumin (BSA) in 0.1% Triton X-100 for 30 min. Primary antibodies were incubated in 1% BSA in 0.1% Triton X-100 overnight at 4 °C at the following concentrations: rabbit polyclonal against TBCA (1:50), TBCB (1:100), TBCC (1:200), TBCD (1:100) and TBCE (1:100, all Proteintech), mouse monoclonal against tyrosinated tubulin (1:500, Millipore Merck), rabbit polyclonal against detyrosinated tubulin (1:500, Millipore Merck), mouse monoclonal against acetylated tubulin (1:500, Sigma-Aldrich) or mouse monoclonal against polyglutamylated tubulin (1:500, AdipoGen).

Techniques: Expressing, Staining, Membrane

Journal: Histochemistry and Cell Biology

Article Title: Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice

doi: 10.1007/s00418-020-01905-6

Figure Lengend Snippet: Distribution of TBC proteins in the organ of Corti at P14. a TBCA labelling is found in both the tectal and Hensen’s cells. β-Tubulin stains the phalangeal extension of the Deiters’ cells. b TBCB expression is detected in basal cell half of the Deiters’ cells, the phalangeal processes are stained by β-tubulin. c The expression of TBCC is found in the apical part of the inner pillar cell, β-tubulin in the Deiters’ cells. d Staining of TBCD is visible in the cell body of outer hair cells. Phalloidin stains the cuticular plate and stereocilia of the outer hair cells and the inner pillar cell. e TBCE is detected in the outer hair cells, whereas Phalloidion marks the cuticular plate and the stereocilia of the outer hair cells and a bundle-like structure extending from the apex to the base of inner pillar cell. (Scale bar = 25 µm)

Article Snippet: For immunofluorescence analysis, sections were post-fixed in 4% PFA for 10 min, rinsed in 0.05 M phosphate-buffered saline three times and blocked in 10% bovine serum albumin (BSA) in 0.1% Triton X-100 for 30 min. Primary antibodies were incubated in 1% BSA in 0.1% Triton X-100 overnight at 4 °C at the following concentrations: rabbit polyclonal against TBCA (1:50), TBCB (1:100), TBCC (1:200), TBCD (1:100) and TBCE (1:100, all Proteintech), mouse monoclonal against tyrosinated tubulin (1:500, Millipore Merck), rabbit polyclonal against detyrosinated tubulin (1:500, Millipore Merck), mouse monoclonal against acetylated tubulin (1:500, Sigma-Aldrich) or mouse monoclonal against polyglutamylated tubulin (1:500, AdipoGen).

Techniques: Expressing, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics

doi: 10.1007/s00018-012-1114-2

Figure Lengend Snippet: TBCB is an autoinhibitory protein. a Schematic drawing of human TBCB depicting the three characterized domains (UBL, coiled-coil and CAP-Gly). The UBL domain (green) corresponds to PDB ID, 1V6E (UBL of murine TBCB), and the CAP-Gly domain (blue) to PDB ID, 1TOV (CAP-Gly domain of F53f4.3) [26]. In light blue are the corresponding residues that form the conserved groove in p150Glued, interacting with the C-terminal peptide of α-tubulin [30, 31]. The last nine residues, which are present in the solved domain of F53f4.3, are shown in red. All structures were drawn using Pymol software (http://www.pymol.org). b Confocal microscopy projection image of TBCBΔ3 overexpression on HeLa cells. TBCBΔ3 (green) produces conspicuous microtubule destruction (white arrow). Moderate TBCBΔ3 levels also severely affect the microtubule cytoskeleton. A high cytoplasmic tubulin background is observed in these two cells. c Statistical analysis of the percentages of cells containing normal, abnormal or absent microtubules in TBCE, TBCB, and TBCBΔ3-overexpressing HeLa cells. A highly significant increase in cells containing a completely destroyed microtubular cytoskeleton is observed when the TBCBΔ3 mutant (asterisk) is overexpressed compared with wild-type TBCB (see also Fig. S1). d The C-terminal region of TBCB functions as an autoinhibitory peptide when bound to the CAP-Gly domain of the protein. The three domains of TBCB are depicted. The N-terminus contains the UBL and the coiled-coil domain, and the C-terminus contains the CAP-Gly domain. The acidic tail of the CAP-Gly domain is shown in red and orange. We propose that the C-terminal tail of TBCB is responsible for the autoinhibition of the protein (red peptide) through interaction with the CAP-Gly domain (blue), specifically with the highly conserved hydrophobic cavity present in the CAP-Gly domain (light blue). In contrast, if the C-terminus region does not interact with the CAP-Gly domain (orange peptide), the protein is derepressed. The hypothetical models of TBCBΔ3 and TBCBΔ9 showing the structure of the protein lacking the last three or nine amino acids are shown

Article Snippet: Human TBCB gene cloning The human Tbcb coding sequence was amplified by PCR from a testis cDNA sample (BD Biosciences, USA) using a pair of primers designed with the appropriate restriction enzyme recognition sites at their ends: forward primer 5′ GTG AAG CTT CAT ATG GAG GTG ACG GGG GTG 3′; reverse primer 5′ CGC GGA TCC TCA TAT CTC GTC CAA CCC 3′.

Techniques: Software, Confocal Microscopy, Over Expression, Mutagenesis

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: The clinical features of AML patients highly expressed TBCB . (A) Transcriptional levels of TBCB in thirty-three different cancers. Data was originated from TCGA and GTEx database. (B) The TBCB mRNA levels of AML patients in comparison with normal subjects. Data was originated from TCGA-LAML and GTEx database. (C-D) The differentially expression of TBCB between AML patients and normal subjects from GSE9476 (C) and GSE13159 (D). (E) RT-qPCR analysis for transcriptional levels of TBCB in BMMNC from AML patients (n = 9) relative to healthy donors (n = 14). Expression levels are normalized to 18S. (F) ROC analysis of TBCB in TCGA-LAML and GTEx datasets. (G-J) Significant clinical features were demonstrated, including count of white blood cells (G), proportions of PB blasts (H) and BM blasts (I), mutation rate of FLT3 (J). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Comparison, Expressing, Quantitative RT-PCR, Mutagenesis

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: Clinical characteristics with diverse TBCB expression levels in AML patients

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Expressing, Mutagenesis

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: High TBCB expression correlated with unfavorable prognosis. (A) Kaplan-Meier survival curve of OS was delineated for AML patients grouped into high versus low expressed populations in line accordance with the median expression of TBCB . Data was originated from TCGA-LAML dataset. (B) Validation of OS for TBCB in the entirely independent cohort GSE37642 (n = 136). (C-D) Forest plots of OS for AML patients from univariate (C) and multivariable (D) analysis. (E) Nomogram based on integrating TBCB and other meaningful prognostic factors of AML. (F) Calibration of the nomogram. (G-I) The DCA curves of the nomogram at 1 year (G), 2 years (H), and 3 years (I).

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Expressing, Biomarker Discovery

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: DEGs and their functional pathways enrichment analysis. (A) Volcano plot showing TBCB -related DEGs, |log 2 FC| ≥ 0.59, p -adjust< 0.05. (B) The bar diagrams display the top five terms for each GO category and KEGG analysis of the up-regulated DEGs, including biological processes (left), cellular components (medium), and KEGG pathways (right). (C-D) Bubble plot (C) and chord plot (D) showing the top 3 BP terms. (E) The first 10 gene sets of GSEA analysis using the Reactome of C2 in MSigDB database. Immune-related gene sets were marked with red. (F) GSEA analysis of immune-related gene sets in up-regulated DEGs.

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Functional Assay

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: Establishment of PPI network and the clinical significance of hub genes. (A-C) The top15 hub genes were acquired with PPI network on the base of MCC (A), MNC (B) and EPC (C) algorithms. (D) The Venn diagram shows the overlap among the top 15 genes sorted by the three algorithms. (E) Expression levels of six hub genes (ITGAM, ITGB2, ITGAX, SPI1, TYROBP, CD68) in TCGA-LAML and GTEx database. (F-G) Co-expression heat map (F) and correlation scatter plots (G) of TBCB with six hub genes. (H-J) The OS in AML patients, splitting into two populations with high versus low expression in the light of the median expression levels of three hub genes, were created by Kaplan-Meier analysis. ITGAM (H), ITGB2 (I), ITGAX (J). * p < 0.05, *** p < 0.001.

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: Correlationships between TBCB expression level and infiltrating immune cells, immune checkpoint in AML. (A) The relationship between the mRNA level of TBCB and twenty four infiltrating immune cells were examined by Spearman's correlation. (B) Correlation scatter plots for nine immune infiltrating cells positively linked to TBCB transcription level. (C-D) Co-expression heat map (C) and correlation scatter plots (D) of TBCB gene with six momentous immune checkpoint molecules in AML. (E) Expression levels of ligands for inhibitory NK cell receptors ( HLA-E , HLA-G and LGALS9 ) in AML. Data was originated from TCGA-LAML and GTEx database. (F) Correlation scatter plots of TBCB gene with three ligands for inhibitory NK cell receptors in AML. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: UP-regulated DEGs with high versus low TBCB expression involved in cell proliferation and apoptosis gene sets in AML patient. (A) Chord plot showing the relevant GO terms of cell proliferation and apoptosis gene sets. (B-C) The Kaplan-Meier survival curves of OS (B) and correlation analysis with TBCB expression (C) for nine genes related to cell proliferation and apoptosis.

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: The effects of TBCB knockdown on cell proliferation in AML human cell lines. (A) RT-qPCR analysis for transcriptional levels of TBCB in AML cell line THP1 (left) and Kasumi-1 (right) transduced with NC-siRNA or siTBCB oligonucleotides. Expression levels are normalized to 18S. (B) Western blotting for TBCB expression in THP1 (left) and Kasumi-1 (right) cell lines transduced with NC-siRNA or siTBCB oligonucleotides. (C-D) Proliferation curves of control and siTBCB groups in THP1 (C) and Kasumi-1 (D) cell lines were measured by CCK8. (E-H) The apoptosis analysis of AML cell lines transfected with siTBCB and NC-siRNA. Representative flow cytometry plots of apoptotic ratio in THP1 (E) and Kasumi-1 (G) cell lines. Statistical analysis of apoptotic ratio in THP1 (F) and Kasumi-1 (H) cell lines. (I-L) The cell cycle analysis of AML cell lines transfected with siTBCB and NC-siRNA. Representative flow cytometric analysis of cell cycle in THP1 (I) and Kasumi-1 (K) cell lines. Quantification of G0, G1, S, G2 and M phases in THP1 (J) and Kasumi-1 (L) cell lines. All statistical values were presented as the means ± SEM. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, by one-way ANOVA.

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Knockdown, Quantitative RT-PCR, Transduction, Expressing, Western Blot, Control, Transfection, Flow Cytometry, Cell Cycle Assay

Journal: Journal of Cancer

Article Title: High Expression of Microtubule-associated Protein TBCB Predicts Adverse Outcome and Immunosuppression in Acute Myeloid Leukemia

doi: 10.7150/jca.84215

Figure Lengend Snippet: Prediction of drug sensitivity based on TBCB expression. (A) CCT018159. (B) 17-AAG. (C) JNJ-268541653. (D) OSU-03012. (E) AG-0140699. (F) Talazoparib. (G) CP724714. (H) Erlotinib. (I) MLN4924. (J) VX-11e. (K) ATRA. (L) Midostaurin. (M) Cytarabine. (N) Sorafenib. (O) Doxorubicin. * p < 0.05, * p < 0.01, *** p < 0.001.

Article Snippet: The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd (Suzhou, China) and transfected into AML cell lines by LipofectamineTM 3000 transfection kit (L3000015, Invitrogen, USA).

Techniques: Expressing