Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rh2 reduces m6A RNA methylation in cancer via the KIF26B-SRF positive feedback loop

doi: 10.1016/j.jgr.2021.05.004

Figure Lengend Snippet: Ginsenoside Rh2 reduces m6A RNA methylation and KIF26B expression in some cancers. A. mRNA m6A content in ginsenoside Rh2 treated (10 or 20 nM, 24 h) cancer cell lines. B. KIF26B mRNA expression in multiple cancer cell lines (the same as in panel A) after treatment with ginsenoside Rh2 (20 nM, 24 h). C-D. Basal KIF26B protein expression in cancer cell lines as in panel A (C) and in three selected cancer cell lines (MDA-MB-157, NMFH-1, and TT) after treatment with ginsenoside Rh2 (20 nM, 24 h) (D). E-F. KIF26B mRNA (top panel) and protein (bottom panel) expression in MDA-MB-157 (E) and NMFH-1 (F) cells with lentiviral mediated KIF26B knockdown, or in combination with lentiviral KIF26B expression vector resistant to shKIF26B#1 (MT-KIF26B). G-J. mRNA m6A content in KIF26B knockdown alone or control MDA-MB-157 (G) and NMFH-1 (H) cells, and in MDA-MB-157 (I) and NMFH-1 (J) cells with ginsenoside Rh2 treatment alone (20 nM, 24 h) or in combination with WT-KIF26B overexpression.

Article Snippet: The promoter sequence of KIF26B was acquired from the KIF26B promoter clone in GeneCopoeia (ID: HPRM49076) ( ).

Techniques: Methylation, Expressing, Knockdown, Plasmid Preparation, Control, Over Expression

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rh2 reduces m6A RNA methylation in cancer via the KIF26B-SRF positive feedback loop

doi: 10.1016/j.jgr.2021.05.004

Figure Lengend Snippet: KIF26B interacts with ZC3H13 and CBLL1 in the cytoplasmic fraction of multiple cancer cell lines. A. IF staining of KIF26 (red), CBLL1 (green), and ZC3H13 (green) in MDA-MB-157, NMFH-1 and TT cells. B. Left panel, Co-IP was conducted using the cytoplasmic fraction MDA-MB-157 (top), NMFH-1 (middle), and TT (bottom) cells. Right panel, reciprocal IP assay was performed to explore the interaction of ZC3H13 and CBLL1 with KIF26B. KIF26B was immunoprecipitated by anti-CBLL1 and anti-C3H13. C and D. The structure of the wild-type full length (FL) and mutation constructs of Flag-tag labeled ZC3H13 (C) and CBLL1 (D). E and F. Flag-tagged FL or truncated mutation constructs were co-transfected with HA-KIF26B into NMFH-1 cells. The KIF26B/Flag-tag complexes were immunoprecipitated by anti-HA antibodies. Flag-ZC3H13 (E) and Flag-CBLL1 (F) proteins were detected using anti-Flag antibodies.

Article Snippet: The promoter sequence of KIF26B was acquired from the KIF26B promoter clone in GeneCopoeia (ID: HPRM49076) ( ).

Techniques: Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Mutagenesis, Construct, FLAG-tag, Labeling, Transfection

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rh2 reduces m6A RNA methylation in cancer via the KIF26B-SRF positive feedback loop

doi: 10.1016/j.jgr.2021.05.004

Figure Lengend Snippet: KIF26B enhances nuclear localization of ZC3H13/CBLL1. A-B. CBLL1 and ZC3H1 3 mRNA expression in MDA-MB-157 (A) and NMFH-1 (B) cells with lentiviral mediated KIF26B knockdown. C–F. KIF26B, ZC3H13, and CBLL1 protein expression in the cytoplasmic (C), and nuclear (N) fractions from MDA-MB-157 (panel C and E) and NMFH-1 (panel D and F) cells with KIF26B knockdown alone (C-D) or in combination with WT-KIF26B overexpression (E-F). G. Immunofluorescence analysis of CBLL1 (green), ZC3H13 (green), and DAPI (blue, cell nuclei) in KIF26B knockdown and control MDA-MB-157 and NMFH-1 cells. Scale bar, 20 μm. H–I. METTL3, METTL14, VIRILIZER, and WTAP protein expression in cytoplasmic (C), and nuclear (N) fractions from MDA-MB-157 (H) and NMFH-1 (I) cells with or without KIF26B knockdown. J-K. Interactions between WTAP and METTL3, METLL14, VIRILIZER, CBLL1, and ZC3H13 were determined by co-IP using cytoplasmic fractions from MDA-MB-157 (J) and NMFH-1 (K) cells with or without KIF26B knockdown. n.s., not significant. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: The promoter sequence of KIF26B was acquired from the KIF26B promoter clone in GeneCopoeia (ID: HPRM49076) ( ).

Techniques: Expressing, Knockdown, Over Expression, Immunofluorescence, Control, Co-Immunoprecipitation Assay

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rh2 reduces m6A RNA methylation in cancer via the KIF26B-SRF positive feedback loop

doi: 10.1016/j.jgr.2021.05.004

Figure Lengend Snippet: SRF activates KIF26B expression via binding to its promoter. A-B. The screening processes and criteria to identify potential TFs (N = 62) and their correlation with KIF26B expression in 60 UPS/MFS cases and 139 BLBC cases in TCGA. C. Predicted binding sites of SRF in the promoter region of KIF26B . D-F. SRF and KIF26B mRNA (D-E) and protein (F) expression in MDA-MB-157 and NMFH-1 cells with lentiviral mediated SRF knockdown. G. The structure of pGL3-KIF26B-promoter-WT and pGL3-KIF26B-promoter-MT plasmid for dual-luciferase assay. H–I. The promoter activity of the WT and MT KIF26B promoter sequence was measured using a dual-luciferase reporter assay. MDA-MB-157 (H) and NMFH-1 (I) cells with or without lentiviral mediated SRF inhibition were co-transfected with pGL3-KIF26B-promoter-WT or pGL3-KIF26B-promoter-MT. J. The positions of primers for ChIP-qPCR assay. K-L. ChIP-qPCR assays were performed using anti-SRF and control IgG antibodies in MDA-MB-157 (K) and NMFH-1 (L) cells. The relative enrichment was presented as % input. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: The promoter sequence of KIF26B was acquired from the KIF26B promoter clone in GeneCopoeia (ID: HPRM49076) ( ).

Techniques: Expressing, Binding Assay, Knockdown, Plasmid Preparation, Luciferase, Activity Assay, Sequencing, Reporter Assay, Inhibition, Transfection, ChIP-qPCR, Control

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rh2 reduces m6A RNA methylation in cancer via the KIF26B-SRF positive feedback loop

doi: 10.1016/j.jgr.2021.05.004

Figure Lengend Snippet: KIF26B modulates SRF m6A methylation in cancer cells. A. Potential m6A modification sites in the sequence of SRF mRNA (NM_003131.3). B–C. Gene-specific m6A RIP-qPCR assay showing the reduction of m6A modification in specific regions of SRF mRNA by shKIF26B in MDA-MB-157 (B) and NMFH-1 (C) cells. D-F. SRF mRNA (D-E) and protein (F) expression in MDA-MB-157 and NMFH-1 cells with lentiviral mediated KIF26B knockdown. G-H. SRF mRNA expression in MDA-MB-157 (G) and NMFH-1 (H) cells at the indicated time points after actinomycin D treatment. I. A potential positive feedback loop between KIF26B and SRF in cancer cells and the regulatory effect of ginsenoside Rh2 on m6A RNA methylation via KIF26B. ∗, Scramble vs. shKIF26B#1; #, Scramble vs. shKIF26B#2; ## and ∗∗, p < 0.01; ### and ∗∗∗, p < 0.001.

Article Snippet: The promoter sequence of KIF26B was acquired from the KIF26B promoter clone in GeneCopoeia (ID: HPRM49076) ( ).

Techniques: Methylation, Modification, Sequencing, Expressing, Knockdown

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rh2 reduces m6A RNA methylation in cancer via the KIF26B-SRF positive feedback loop

doi: 10.1016/j.jgr.2021.05.004

Figure Lengend Snippet: SRF-KIF26B axis enhances the growth of cancer cells in vitro and in vi vo. A-D. Cell proliferation of MDA-MB-157 (A) and NMFH-1 (B) cells with SRF or KIF26B inhibition alone (A-B) or combined SRF inhibition and KIF26B overexpression (C-D). E-H. Representative images (E and G) and statistical analysis (F and H) of colony formation of MDA-MB-157 (E-F) and NMFH-1 (G-H) cells with SRF or KIF26B inhibition alone or combined SRF inhibition and KIF26B overexpression. I-L. Representative images of MDA-MB-157 (I-J) and NMFH-1 (K-L) cell (with SRF or KIF26B inhibition) derived xenograft tumors (I and K) and the growth curves (J and L). M-T. Kaplan-Meier survival analysis of RFS and OS in TNBC cases from the Kaplan Meier plotter (M-P) and PFS and OS in UPS/MFS cases in TCGA-SRAC (Q-T). ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: The promoter sequence of KIF26B was acquired from the KIF26B promoter clone in GeneCopoeia (ID: HPRM49076) ( ).

Techniques: In Vitro, Inhibition, Over Expression, Derivative Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Schematic overview of the study design investigating the role of KIF26B in paclitaxel resistance and prognosis of ovarian cancer.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated overnight at 4 °C with an anti-KIF26B antibody (1:750 dilution, Proteintech, Rosemont, IL, USA, #17422-1-AP).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: KIF26B expression in ovarian cancer and its correlation with clinical stage and prognosis. ( A ) KIF26B mRNA expression levels in 88 normal ovarian tissues (Normal) and 426 ovarian cancer tissues (Tumor) based on RNA-seq data from TCGA and GTEx datasets. (* p < 0.05). ( B ) KIF26B mRNA expression levels in 20 stage II ovarian cancer tissues (Stage 2), 243 stage III ovarian cancer tissues (Stage 3), and 38 stage IV ovarian cancer tissues (Stage 4) derived from TCGA ovarian cancer cohort. (* p < 0.05, *** p < 0.001). ( C , D ) Protein expression levels of KIF26B in ovarian cancer tissues were assessed using four independent antibodies (HPA027709, HPA028478, HPA028561, HPA028562) from the Human Protein Atlas database. The representative IHC images of four expression levels (not detected, low, medium, high) are displayed alongside a column graph quantifying the number of patients at each expression level per antibody. ( E ) Kaplan-Meier analysis was performed to evaluate the association of KIF26B expression with OS, PFS and PPS in patients with ovarian cancer. The KIF26B level was classified as low (L) and high (H) using the auto-selected best cut-off value. OS, overall survival; PFS, progression-free survival; PPS, postprogression survival.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated overnight at 4 °C with an anti-KIF26B antibody (1:750 dilution, Proteintech, Rosemont, IL, USA, #17422-1-AP).

Techniques: Expressing, RNA Sequencing, Derivative Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: KIF26B expression in chemoresistant ovarian cancer tissues and the prediction of therapeutic response. Using a large sample of 1347 ovarian cancer cases, ROC plotter was employed to analyse KIF26B expression in chemoresistant (Resistance) and chemosensitive (Sensitive) tissues, as well as its correlation with chemotherapy resistance prediction in ovarian cancer. “Any drugs” refers to all chemotherapy drugs; “Taxane” refers to paclitaxel; “Platin” refers to platinum-based drugs; “Serous” refers to serous ovarian cancer; “Stage 3” refers to stage III ovarian cancer; and “Grade 3” refers to poorly differentiated grade III ovarian cancer. “Taxane-serous-Stage 3-Grade 3” indicates tissues from patients with poorly differentiated stage III serous ovarian cancer treated with paclitaxel, and a similar classification applies to other patients. The classification of chemoresistant and chemosensitive tissues is based on whether ovarian cancer recurred within 6 months of chemotherapy.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated overnight at 4 °C with an anti-KIF26B antibody (1:750 dilution, Proteintech, Rosemont, IL, USA, #17422-1-AP).

Techniques: Expressing, Clinical Proteomics

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: KIF26B knockdown inhibits ovarian cancer cell proliferation. ( A ) RT-qPCR analysis of KIF26B mRNA expression levels in H and H-R cells, *** p < 0.001. ( B ) Western blotting analysis of KIF26B protein expression levels in H and H-R cells, *** p < 0.001. H: the parent cell HeyA8; H-R: the paclitaxel-resistant ovarian cancer cell HeyA8-R. ( C ) RT-qPCR analysis of KIF26B mRNA expression levels in four cell lines, i.e., H-Scramble, H-shKIF26B, H-R-Scramble, and H-R-shKIF26B cells, *** p < 0.001. ( D ) Western blotting analysis of KIF26B protein expression levels in the same four cell lines, i.e., H-Scramble, H-shKIF26B, H-R-Scramble, and H-R-shKIF26B cells, ** p < 0.01, *** p < 0.001. ( E , F ) Seven-day growth curves of ( E ) H-Scramble/H-shKIF26B cells and ( F ) H-R-Scramble/H-R-shKIF26B cells. Cell proliferation was measured by the CCK-8 assay. The experiments were conducted in triplicate, and comparisons between scramble cells and shKIF26B cells treated with the same paclitaxel concentration were performed using two-way ANOVA; ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated overnight at 4 °C with an anti-KIF26B antibody (1:750 dilution, Proteintech, Rosemont, IL, USA, #17422-1-AP).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Concentration Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Effects of KIF26B knockdown on paclitaxel resistance in ovarian cancer cells. ( A , B ) Dose-dependent inhibition curves of ( A ) H-Scramble/H-shKIF26B cells and ( B ) H-R-Scramble/H-R-shKIF26B cells after 72 h of treatment with increasing concentrations of paclitaxel. In both panels, the black curve represents the Scramble control group, and the red curve represents the shKIF26B group. The IC 50 values of paclitaxel in these two transfected cell lines are compared, ** p < 0.01, *** p < 0.001. ( C , D ) Scanned images of crystal violet-stained cells from the colony formation assay after 9 days of treatment with varying doses of paclitaxel in ( C ) H-Scramble/H-shKIF26B cells and ( D ) H-R-Scramble/H-R-shKIF26B cells. Relative colony formation efficiency was quantified using ImageJ software on the basis of the colony formation results, * p < 0.05, *** p < 0.001. ( E , F ) Representative confocal images of tubulin immunofluorescence staining following 72 h of paclitaxel treatment in ( E ) H-Scramble/H-shKIF26B cells and ( F ) H-R-Scramble and H-R-shKIF26B cells. Red fluorescence indicates α-tubulin. DAPI was used to stain nuclei. The “Merge” image shows the combined staining of α-tubulin and DAPI.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated overnight at 4 °C with an anti-KIF26B antibody (1:750 dilution, Proteintech, Rosemont, IL, USA, #17422-1-AP).

Techniques: Knockdown, Inhibition, Control, Transfection, Staining, Colony Assay, Software, Immunofluorescence, Fluorescence

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Effects of KIF26B knockdown on cell cycle distribution and apoptosis in response to paclitaxel treatment. ( A , B ) Cell cycle distribution analysed by flow cytometry and ModFit after 72 h of treatment with increasing concentrations of paclitaxel in ( A ) H-Scramble/H-shKIF26B cells and ( B ) H-R-Scramble/H-R-shKIF26B cells. In the flow cytometry histograms, red regions correspond to the populations of cells in the G 0 /G 1 phase (left peak, 2N DNA content) and the G 2 /M phase (right peak, 4N DNA content); purple regions represent cells in the S phase (DNA content between 2N and 4N). The overlaid black line represents the fitted curve. Quantitative analysis of the percentage of cells in the G 0 /G 1 , S, and G 2 /M phases after exposure to varying paclitaxel concentrations for 72 h. ( C , D ) Representative flow cytometry plots of apoptotic ( C ) H-Scramble/H-shKIF26B cells and ( D ) H-R-Scramble/H-R-shKIF26B cells after 72 h of paclitaxel exposure, as assessed by PE Annexin V/7-AAD double staining; quantification of apoptosis rates, including viable, early apoptotic, late apoptotic, and total apoptotic populations. All experiments were repeated three times, and statistical comparisons between scramble cells and shKIF26B cells at the same paclitaxel concentration were performed using two-way ANOVA. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated overnight at 4 °C with an anti-KIF26B antibody (1:750 dilution, Proteintech, Rosemont, IL, USA, #17422-1-AP).

Techniques: Knockdown, Flow Cytometry, Double Staining, Concentration Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Expression patterns of KIF26B and SLC7A11 in ovarian cancer and their associations with prognosis and paclitaxel response. ( A ) Analysis of the correlation between KIF26B and SLC7A11 expression in 489 ovarian cancer tissues derived from TCGA transcriptome data. ( B ) Combined analysis of KIF26B and SLC7A11 expression in ovarian cancer patients revealed significant differences in OS, PFS and PPS between the combined expression groups: a (L + H), b (H + H), c (L + L), and d (H + L). OS, overall survival; PFS, progression-free survival; PPS, post-progression survival. ( C , D ) Western blotting analysis of SLC7A11 protein levels in ( C ) H-Scramble/H-shKIF26B cells and ( D ) H-R-Scramble/H-R-shKIF26B cells. Data were normalized to β-actin and are presented as mean ± SD of three independent experiments, ** p < 0.01. ( E ) Dose-dependent inhibition curves and IC 50 values of paclitaxel in H-R cells with combinatorial knockdown of KIF26B and SLC7A11. Cells with stable KIF26B knockdown or control (Scramble) stable lines were transfected with siRNA targeting SLC7A11 or a non-targeting control (si-NC). Groups a-d were then treated with a paclitaxel gradient for 72 h. Cell inhibition rate was quantified using the CellTiter-Glo luminescent assay. Data points (mean ± SD of three independent experiments) represent the dose-dependent inhibition, and the corresponding IC 50 are displayed within the panel. Group definitions: a, H-R-Scramble + si-NC; b, H-R-Scramble + si-SLC7A11 A; c, H-R-shKIF26B + si-NC; d, H-R-shKIF26B + si-SLC7A11.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated overnight at 4 °C with an anti-KIF26B antibody (1:750 dilution, Proteintech, Rosemont, IL, USA, #17422-1-AP).

Techniques: Expressing, Derivative Assay, Western Blot, Inhibition, Knockdown, Control, Transfection, Luminescence Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Protein-protein docking analysis of the interaction between KIF26B and SLC7A11. ( A ) Predicted three-dimensional docking model of the KIF26B-SLC7A11 complex generated using the HDOCK server. KIF26B is shown in purple, and SLC7A11 is shown in cyan. The putative interaction interface is highlighted. ( B ) Enlarged view of the predicted interaction interface, highlighting residues from KIF26B-SLC7A11 that are implicated in intermolecular contacts. Putative hydrogen bonds and salt bridge interactions are indicated by dashed lines. ( C ) Two-dimensional schematic representation of the predicted intermolecular interactions between KIF26B-SLC7A11 generated using LigPlot+ version 2.2.4. Putative hydrogen bonds and salt bridge interactions are indicated by dashed lines, while hydrophobic contacts are shown as red arcs. Residues at the predicted interaction interface are labeled.

Article Snippet: After blocking with 5% nonfat milk in TBST, the membranes were incubated overnight at 4 °C with an anti-KIF26B antibody (1:750 dilution, Proteintech, Rosemont, IL, USA, #17422-1-AP).

Techniques: Generated, Labeling

Journal: Medical Science Monitor

Article Title: Upregulation of KIF26B, Cell Migration and Proliferation of Human Ovarian Cancer Cell Lines In Vitro, and Patient Outcomes from Human Bioinformatic Analysis

doi: 10.12659/msm.907889

Figure Lengend Snippet: Figure 1. The KIF26B gene is not expressed in normal ovarian tissues, but is upregulated in ovarian cancer. (A, B) The expression of

Article Snippet: Lentiviral KIF26B shRNA was obtained from OriGene (Rockville, MD, USA).

Techniques: Expressing

Journal: Medical Science Monitor

Article Title: Upregulation of KIF26B, Cell Migration and Proliferation of Human Ovarian Cancer Cell Lines In Vitro, and Patient Outcomes from Human Bioinformatic Analysis

doi: 10.12659/msm.907889

Figure Lengend Snippet: Figure 4. High KIF26B gene expression is associated with poor overall survival (OS), progression-free survival (PFS), and post-

Article Snippet: Lentiviral KIF26B shRNA was obtained from OriGene (Rockville, MD, USA).

Techniques: Gene Expression

Journal: Medical Science Monitor

Article Title: Upregulation of KIF26B, Cell Migration and Proliferation of Human Ovarian Cancer Cell Lines In Vitro, and Patient Outcomes from Human Bioinformatic Analysis

doi: 10.12659/msm.907889

Figure Lengend Snippet: Figure 5. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of KIF26B and its co-expressed genes in The Cancer

Article Snippet: Lentiviral KIF26B shRNA was obtained from OriGene (Rockville, MD, USA).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Schematic overview of the study design investigating the role of KIF26B in paclitaxel resistance and prognosis of ovarian cancer.

Article Snippet: To create stable cell lines with KIF26B knockdown, lentiviral particles targeting the KIF26B gene provided by Cyagen (Santa Clara, CA, USA) were used.

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: KIF26B expression in ovarian cancer and its correlation with clinical stage and prognosis. ( A ) KIF26B mRNA expression levels in 88 normal ovarian tissues (Normal) and 426 ovarian cancer tissues (Tumor) based on RNA-seq data from TCGA and GTEx datasets. (* p < 0.05). ( B ) KIF26B mRNA expression levels in 20 stage II ovarian cancer tissues (Stage 2), 243 stage III ovarian cancer tissues (Stage 3), and 38 stage IV ovarian cancer tissues (Stage 4) derived from TCGA ovarian cancer cohort. (* p < 0.05, *** p < 0.001). ( C , D ) Protein expression levels of KIF26B in ovarian cancer tissues were assessed using four independent antibodies (HPA027709, HPA028478, HPA028561, HPA028562) from the Human Protein Atlas database. The representative IHC images of four expression levels (not detected, low, medium, high) are displayed alongside a column graph quantifying the number of patients at each expression level per antibody. ( E ) Kaplan-Meier analysis was performed to evaluate the association of KIF26B expression with OS, PFS and PPS in patients with ovarian cancer. The KIF26B level was classified as low (L) and high (H) using the auto-selected best cut-off value. OS, overall survival; PFS, progression-free survival; PPS, postprogression survival.

Article Snippet: To create stable cell lines with KIF26B knockdown, lentiviral particles targeting the KIF26B gene provided by Cyagen (Santa Clara, CA, USA) were used.

Techniques: Expressing, RNA Sequencing, Derivative Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: KIF26B expression in chemoresistant ovarian cancer tissues and the prediction of therapeutic response. Using a large sample of 1347 ovarian cancer cases, ROC plotter was employed to analyse KIF26B expression in chemoresistant (Resistance) and chemosensitive (Sensitive) tissues, as well as its correlation with chemotherapy resistance prediction in ovarian cancer. “Any drugs” refers to all chemotherapy drugs; “Taxane” refers to paclitaxel; “Platin” refers to platinum-based drugs; “Serous” refers to serous ovarian cancer; “Stage 3” refers to stage III ovarian cancer; and “Grade 3” refers to poorly differentiated grade III ovarian cancer. “Taxane-serous-Stage 3-Grade 3” indicates tissues from patients with poorly differentiated stage III serous ovarian cancer treated with paclitaxel, and a similar classification applies to other patients. The classification of chemoresistant and chemosensitive tissues is based on whether ovarian cancer recurred within 6 months of chemotherapy.

Article Snippet: To create stable cell lines with KIF26B knockdown, lentiviral particles targeting the KIF26B gene provided by Cyagen (Santa Clara, CA, USA) were used.

Techniques: Expressing, Clinical Proteomics

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: KIF26B knockdown inhibits ovarian cancer cell proliferation. ( A ) RT-qPCR analysis of KIF26B mRNA expression levels in H and H-R cells, *** p < 0.001. ( B ) Western blotting analysis of KIF26B protein expression levels in H and H-R cells, *** p < 0.001. H: the parent cell HeyA8; H-R: the paclitaxel-resistant ovarian cancer cell HeyA8-R. ( C ) RT-qPCR analysis of KIF26B mRNA expression levels in four cell lines, i.e., H-Scramble, H-shKIF26B, H-R-Scramble, and H-R-shKIF26B cells, *** p < 0.001. ( D ) Western blotting analysis of KIF26B protein expression levels in the same four cell lines, i.e., H-Scramble, H-shKIF26B, H-R-Scramble, and H-R-shKIF26B cells, ** p < 0.01, *** p < 0.001. ( E , F ) Seven-day growth curves of ( E ) H-Scramble/H-shKIF26B cells and ( F ) H-R-Scramble/H-R-shKIF26B cells. Cell proliferation was measured by the CCK-8 assay. The experiments were conducted in triplicate, and comparisons between scramble cells and shKIF26B cells treated with the same paclitaxel concentration were performed using two-way ANOVA; ** p < 0.01, *** p < 0.001.

Article Snippet: To create stable cell lines with KIF26B knockdown, lentiviral particles targeting the KIF26B gene provided by Cyagen (Santa Clara, CA, USA) were used.

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Concentration Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Effects of KIF26B knockdown on paclitaxel resistance in ovarian cancer cells. ( A , B ) Dose-dependent inhibition curves of ( A ) H-Scramble/H-shKIF26B cells and ( B ) H-R-Scramble/H-R-shKIF26B cells after 72 h of treatment with increasing concentrations of paclitaxel. In both panels, the black curve represents the Scramble control group, and the red curve represents the shKIF26B group. The IC 50 values of paclitaxel in these two transfected cell lines are compared, ** p < 0.01, *** p < 0.001. ( C , D ) Scanned images of crystal violet-stained cells from the colony formation assay after 9 days of treatment with varying doses of paclitaxel in ( C ) H-Scramble/H-shKIF26B cells and ( D ) H-R-Scramble/H-R-shKIF26B cells. Relative colony formation efficiency was quantified using ImageJ software on the basis of the colony formation results, * p < 0.05, *** p < 0.001. ( E , F ) Representative confocal images of tubulin immunofluorescence staining following 72 h of paclitaxel treatment in ( E ) H-Scramble/H-shKIF26B cells and ( F ) H-R-Scramble and H-R-shKIF26B cells. Red fluorescence indicates α-tubulin. DAPI was used to stain nuclei. The “Merge” image shows the combined staining of α-tubulin and DAPI.

Article Snippet: To create stable cell lines with KIF26B knockdown, lentiviral particles targeting the KIF26B gene provided by Cyagen (Santa Clara, CA, USA) were used.

Techniques: Knockdown, Inhibition, Control, Transfection, Staining, Colony Assay, Software, Immunofluorescence, Fluorescence

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Effects of KIF26B knockdown on cell cycle distribution and apoptosis in response to paclitaxel treatment. ( A , B ) Cell cycle distribution analysed by flow cytometry and ModFit after 72 h of treatment with increasing concentrations of paclitaxel in ( A ) H-Scramble/H-shKIF26B cells and ( B ) H-R-Scramble/H-R-shKIF26B cells. In the flow cytometry histograms, red regions correspond to the populations of cells in the G 0 /G 1 phase (left peak, 2N DNA content) and the G 2 /M phase (right peak, 4N DNA content); purple regions represent cells in the S phase (DNA content between 2N and 4N). The overlaid black line represents the fitted curve. Quantitative analysis of the percentage of cells in the G 0 /G 1 , S, and G 2 /M phases after exposure to varying paclitaxel concentrations for 72 h. ( C , D ) Representative flow cytometry plots of apoptotic ( C ) H-Scramble/H-shKIF26B cells and ( D ) H-R-Scramble/H-R-shKIF26B cells after 72 h of paclitaxel exposure, as assessed by PE Annexin V/7-AAD double staining; quantification of apoptosis rates, including viable, early apoptotic, late apoptotic, and total apoptotic populations. All experiments were repeated three times, and statistical comparisons between scramble cells and shKIF26B cells at the same paclitaxel concentration were performed using two-way ANOVA. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To create stable cell lines with KIF26B knockdown, lentiviral particles targeting the KIF26B gene provided by Cyagen (Santa Clara, CA, USA) were used.

Techniques: Knockdown, Flow Cytometry, Double Staining, Concentration Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Expression patterns of KIF26B and SLC7A11 in ovarian cancer and their associations with prognosis and paclitaxel response. ( A ) Analysis of the correlation between KIF26B and SLC7A11 expression in 489 ovarian cancer tissues derived from TCGA transcriptome data. ( B ) Combined analysis of KIF26B and SLC7A11 expression in ovarian cancer patients revealed significant differences in OS, PFS and PPS between the combined expression groups: a (L + H), b (H + H), c (L + L), and d (H + L). OS, overall survival; PFS, progression-free survival; PPS, post-progression survival. ( C , D ) Western blotting analysis of SLC7A11 protein levels in ( C ) H-Scramble/H-shKIF26B cells and ( D ) H-R-Scramble/H-R-shKIF26B cells. Data were normalized to β-actin and are presented as mean ± SD of three independent experiments, ** p < 0.01. ( E ) Dose-dependent inhibition curves and IC 50 values of paclitaxel in H-R cells with combinatorial knockdown of KIF26B and SLC7A11. Cells with stable KIF26B knockdown or control (Scramble) stable lines were transfected with siRNA targeting SLC7A11 or a non-targeting control (si-NC). Groups a-d were then treated with a paclitaxel gradient for 72 h. Cell inhibition rate was quantified using the CellTiter-Glo luminescent assay. Data points (mean ± SD of three independent experiments) represent the dose-dependent inhibition, and the corresponding IC 50 are displayed within the panel. Group definitions: a, H-R-Scramble + si-NC; b, H-R-Scramble + si-SLC7A11 A; c, H-R-shKIF26B + si-NC; d, H-R-shKIF26B + si-SLC7A11.

Article Snippet: To create stable cell lines with KIF26B knockdown, lentiviral particles targeting the KIF26B gene provided by Cyagen (Santa Clara, CA, USA) were used.

Techniques: Expressing, Derivative Assay, Western Blot, Inhibition, Knockdown, Control, Transfection, Luminescence Assay

Journal: Current Issues in Molecular Biology

Article Title: Low KIF26B Expression Reduces Paclitaxel Resistance and Predicts Good Prognosis in Ovarian Cancer

doi: 10.3390/cimb48020226

Figure Lengend Snippet: Protein-protein docking analysis of the interaction between KIF26B and SLC7A11. ( A ) Predicted three-dimensional docking model of the KIF26B-SLC7A11 complex generated using the HDOCK server. KIF26B is shown in purple, and SLC7A11 is shown in cyan. The putative interaction interface is highlighted. ( B ) Enlarged view of the predicted interaction interface, highlighting residues from KIF26B-SLC7A11 that are implicated in intermolecular contacts. Putative hydrogen bonds and salt bridge interactions are indicated by dashed lines. ( C ) Two-dimensional schematic representation of the predicted intermolecular interactions between KIF26B-SLC7A11 generated using LigPlot+ version 2.2.4. Putative hydrogen bonds and salt bridge interactions are indicated by dashed lines, while hydrophobic contacts are shown as red arcs. Residues at the predicted interaction interface are labeled.

Article Snippet: To create stable cell lines with KIF26B knockdown, lentiviral particles targeting the KIF26B gene provided by Cyagen (Santa Clara, CA, USA) were used.

Techniques: Generated, Labeling

Journal: Cancer Cell International

Article Title: miR-450b-5p loss mediated KIF26B activation promoted hepatocellular carcinoma progression by activating PI3K/AKT pathway

doi: 10.1186/s12935-019-0923-x

Figure Lengend Snippet: Expression and clinical relevance of KIF26B in TCGA cohort of HCC patients. a Expression of KIF26B in HCC tissues and normal tissues. b , c Expression of KIF26B in different TNM stage and cell differentiation of TCGA HCC cohort. d Relationship between expression level of KIF26B and Ki67 in TCGA HCC cohort. e , f Relationship between expression level of KIF26B and overall survival and disease-free survival of TCGA HCC patients. g , h Stratified analyses between TNM stages and KIF26B expression on overall survival and disease-free survival

Article Snippet: The coding sequences of human KIF26B and negative control were amplified and cloned into pcDNA3.1 (+) to generate KIF26B overexpression and according negative control (GenePharma, Shanghai, China). miR-450b-5p mimics, inhibitors as well as respective negative controls (NC) were commercially purchased from Gene Pharma (Shanghai, China).

Techniques: Expressing, Cell Differentiation

Journal: Cancer Cell International

Article Title: miR-450b-5p loss mediated KIF26B activation promoted hepatocellular carcinoma progression by activating PI3K/AKT pathway

doi: 10.1186/s12935-019-0923-x

Figure Lengend Snippet: Suppression of KIF26B inhibits proliferation rate and invasion ability of HCC cells in vitro. a Expression difference of KIF26B between HCC tissues and adjacent non-tumor tissues was showed. b Protein level of KIF26B between normal hepatocytes cell lines and HCC cell lines was compared. And transfect efficiency of KIF26B shRNA in Hep-3B and HCC-LM3 cells was verified in protein level. c – e Cell viability of Hep-3B and HCC-LM3 cells under treatment of KIF26B shRNA and negative control in 24 h, 48 h and 72 h. f , g Cell proliferation rate and invasion ability of Hep-3B and HCC-LM3 cells under treatment of KIF26B shRNA and negative control. h Tumor formation ability of HCC cells with KIF26B shRNA and Negative control was compared in vitro. Mean ± SD, *p < 0.05, unpaired Student’s t test or one-way ANNOVA followed by multiple t test

Article Snippet: The coding sequences of human KIF26B and negative control were amplified and cloned into pcDNA3.1 (+) to generate KIF26B overexpression and according negative control (GenePharma, Shanghai, China). miR-450b-5p mimics, inhibitors as well as respective negative controls (NC) were commercially purchased from Gene Pharma (Shanghai, China).

Techniques: In Vitro, Expressing, shRNA, Negative Control

Journal: Cancer Cell International

Article Title: miR-450b-5p loss mediated KIF26B activation promoted hepatocellular carcinoma progression by activating PI3K/AKT pathway

doi: 10.1186/s12935-019-0923-x

Figure Lengend Snippet: Suppression of KIF26B inhibits tumor formation ability of HCC cells in vivo. a – d The in vivo effect of KIF26B was evaluated in xenograft mouse models bearing tumors originating from HCC-LM3 cells via tumor volume, photon flux and tumor weight; n = 6 per group. e , f Expression of KIF26B and Ki67 were detected between KIF26B shRNA and negative control groups in node mouse tumor tissues

Article Snippet: The coding sequences of human KIF26B and negative control were amplified and cloned into pcDNA3.1 (+) to generate KIF26B overexpression and according negative control (GenePharma, Shanghai, China). miR-450b-5p mimics, inhibitors as well as respective negative controls (NC) were commercially purchased from Gene Pharma (Shanghai, China).

Techniques: In Vivo, Expressing, shRNA, Negative Control

Journal: Cancer Cell International

Article Title: miR-450b-5p loss mediated KIF26B activation promoted hepatocellular carcinoma progression by activating PI3K/AKT pathway

doi: 10.1186/s12935-019-0923-x

Figure Lengend Snippet: KIF26B regulates proliferation and invasion of HCC cells through PI3K/AKT pathway. a , b The GO and KEGG enrichment analysis was conducted in TCGA HCC cohort between KIF26B high expression and KIF26B low expression. c , d The Gene Set Enrichment Analysis plot indicated a significant correlation between KIF26B and PI3K/AKT pathway. e , f Expression change of m-TOR, p-AKT and p-PI3K in HCC cells and xenograft mouse models bearing tumors under condition of KIF26B suppression

Article Snippet: The coding sequences of human KIF26B and negative control were amplified and cloned into pcDNA3.1 (+) to generate KIF26B overexpression and according negative control (GenePharma, Shanghai, China). miR-450b-5p mimics, inhibitors as well as respective negative controls (NC) were commercially purchased from Gene Pharma (Shanghai, China).

Techniques: Expressing

Journal: Cancer Cell International

Article Title: miR-450b-5p loss mediated KIF26B activation promoted hepatocellular carcinoma progression by activating PI3K/AKT pathway

doi: 10.1186/s12935-019-0923-x

Figure Lengend Snippet: KIF26B is targeting regulated by miR-450b-5p in HCC cells. a miR-450b-5p could be specific complementary pairing with 3′UTR sequence of KIF26B. b , c Expression difference of miR-450b-5p between HCC tissues and adjacent non-tumor tissues in our hospital HCC cohort and TCGA HCC cohort. d , e Protein and mRNA change of KIF26B in HCC cells under treatment of miR-450b-5p mimics, inhibitor and negative control. f Relative luciferase activity between KIF26B wild type and mutant type based on a dual-luciferase reporter assay. Values represent the mean ± SD (n = 5, each). g Relationship between KIF26B expression and miR-450b-5p expression in HCC tissues. *p < 0.05

Article Snippet: The coding sequences of human KIF26B and negative control were amplified and cloned into pcDNA3.1 (+) to generate KIF26B overexpression and according negative control (GenePharma, Shanghai, China). miR-450b-5p mimics, inhibitors as well as respective negative controls (NC) were commercially purchased from Gene Pharma (Shanghai, China).

Techniques: Sequencing, Expressing, Negative Control, Luciferase, Activity Assay, Mutagenesis, Reporter Assay

Journal: Cancer Cell International

Article Title: miR-450b-5p loss mediated KIF26B activation promoted hepatocellular carcinoma progression by activating PI3K/AKT pathway

doi: 10.1186/s12935-019-0923-x

Figure Lengend Snippet: Overexpression of KIF26B could reverse tumor suppressive effect of miR-450b-5p in HCC cells. a KIF26B expression under condition of miR-450b-5p mimics, miR-450b-5p mimics and KIF26B or negative control. b Cell viability of Hep-3B and HCC-LM3 cells under treatment of miR-450b-5p mimics, miR-450b-5p mimics and KIF26B and negative control in 24 h, 48 h and 72 h. c , d Cell proliferation rate and invasion ability of Hep-3B and HCC-LM3 cells under treatment of miR-450b-5p mimics, miR-450b-5p mimics and KIF26B and negative control. e Tumor formation ability of HCC cells with miR-450b-5p mimics, miR-450b-5p mimics and KIF26B and negative control was compared in vitro. Mean ± SD, *p < 0.05, unpaired Student’s t test or one-way ANNOVA followed by multiple t test

Article Snippet: The coding sequences of human KIF26B and negative control were amplified and cloned into pcDNA3.1 (+) to generate KIF26B overexpression and according negative control (GenePharma, Shanghai, China). miR-450b-5p mimics, inhibitors as well as respective negative controls (NC) were commercially purchased from Gene Pharma (Shanghai, China).

Techniques: Over Expression, Expressing, Negative Control, In Vitro

Journal: Cancer Cell International

Article Title: miR-450b-5p loss mediated KIF26B activation promoted hepatocellular carcinoma progression by activating PI3K/AKT pathway

doi: 10.1186/s12935-019-0923-x

Figure Lengend Snippet: Negative correlation between KIF26B and miR-450b-5p is associated with prognosis of HCC patients. a , b Relationship between expression level of miR-450b-5p and overall survival and disease-free survival of TCGA HCC patients. c , d Overall survival and disease-free survival were compared among HCC patients with miR-450b-5p low KIF26B low , miR-450b-5p low KIF26B high , miR-450b-5p high KIF26B low and miR-450b-5p high KIF26B high

Article Snippet: The coding sequences of human KIF26B and negative control were amplified and cloned into pcDNA3.1 (+) to generate KIF26B overexpression and according negative control (GenePharma, Shanghai, China). miR-450b-5p mimics, inhibitors as well as respective negative controls (NC) were commercially purchased from Gene Pharma (Shanghai, China).

Techniques: Expressing