Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Peroxisome Deficiency in Cochlear Hair Cells Causes Hearing Loss by Deregulating BK Channels.

doi: 10.1002/advs.202300402

Figure Lengend Snippet: Figure 6. BK channel expression is decreased in cochlear hair cells of Atoh1-Pex5−/−mice. A) Schematic representation of the distribution of several important proteins involved in hair cell function. There are three important proteins in the inner hair cells: Glutamate, GluR, and BK channels, and there are three important proteins in the outer hair cells: PMCA2, Prestin, and KCNQ4. Among them, voltage-gated Ca2+ channels are distributed at the connection between the inner and outer hair cells at the afferent terminal. B) No significant difference was observed in the expression and distribution of PMCA2 between 1-month WT and Atoh1-Pex5−/−mice, n = 3 for each group. Scale bar 10 μm. C) No significant difference in the distribution and expression of Prestin protein in hair cells between 1-month WT and Atoh1-Pex5−/−mice, n = 5 for each group. Scale bar 10 μm. D) Immunofluorescence of KCNQ4 (red) from 1-month WT and Atoh1-Pex5−/−mice, n = 6 for each group. Scale bar 10 μm. E) Western blotting analysis of KCNQ4 and Prestin in the cochlea from 1-month WT and Atoh1-Pex5−/−mice. F) Quantification of KCNQ4 and Prestin levels from (E), n = 3 for each group. G) Immunofluorescence images of the BK 𝛼-subunit of the cochlea sensory epithelium from 1-month WT and Atoh1-Pex5−/- mice. The area selected by the white box is enlarged and displayed on the right side of the image. Scale bar 10 μm. H) BK-channel expression in hair cells of 1-month Atoh1-Pex5−/- mice was significantly reduced in apical, middle, and basal turns compared to 1-month WT mice, n = 3 for each group. Data represent the means ±SEM. “ns” represents not significant, *** p < 0.001, by two-tailed Student’s t-test.

Article Snippet: The following antibodies were used: PMP70 (ab3421, Abcam, 1:200), Parvalbumin (MAB1572, Sigma, 1:2000), Pex5 (NBP1-87185, Novus Biologicals, 1:200), Myo7a (25-6790, Proteus Biosciences, 1:200), Sox2 (ab97959, Abcam, 1:200), NF-200 (ab4680, Abcam, 1:200), 4-HNE (1:100, Abcam, ab46545), P-S6 (Ser 235/236) (4858T, CST, 1:200), PMCA2 (PA1-915, ThermoFisher Scientific, 1:200), Prestin (sc-2294, Santa Cruz, 1:200), KCNQ4 (SMC-309, StressMarq Biosciences, 1:200), KCNMA1 (APC-107, Alomone Labs, 1:200), TOMM20 (ab56783, Abcam, 1:200), otoferlin (ab53233, Abcam, 1:200), Ctbp2 (612 044, BD Transduction Laboratories, 1:200), GluR2 (MAB397, Millipore, 1:200), and Phalloidin-iFluor 594 (ab176757, Abcam, 1:2000).

Techniques: Expressing, Cell Function Assay, Western Blot, Two Tailed Test

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLND4 promotes malignancy in glioma cells. A. Expression levels (mRNA) of CLDN4 in normal tissues (n=5) and glioma tissues (n=156); data derived from TCGA database. B. Representative images of immunohistochemical staining for CLDN4 in para-carcinomatous tissues (n=15), early-stage glioma tissues (stage I-II, n=15), and late-stage glioma tissues (stage III-IV, n=15); scale bar =50 μm. C. Kaplan-Meier survival curve for high and low expression of CLDN4 in glioma patients (n=513); data from TCGA database. D. Western blotting for CLDN4 in LN229/T98G cells transfected with empty vector or a CLDN4-overexpression vector. E. Cell proliferation assay (CCK-8 assay) for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector. T98G, P<0.05, 72 hours; LN229, P<0.01, 72 hours. F. Cell migration assay (Transwell assay) for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector; scale bar =100 μm. G. Colony formation assay for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector.

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining, Western Blot, Transfection, Plasmid Preparation, Over Expression, Proliferation Assay, CCK-8 Assay, Cell Migration Assay, Transwell Assay, Colony Assay

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLDN4-stimulated NNAT upregulation. A. The top 30 upregulated genes in patients with gliomas that had high CLDN4 expression levels as compared to those with gliomas having low CLDN4 expression levels; data derived from TCGA database (n=513). B. Western blotting for CLDN4 and NNAT in LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector. C. Cell proliferation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. LN229, vector vs vector + siRNA, P>0.05, OE vs OE + siRNA, P<0.05, 72 hours; T98G, vector vs vector + siRNA, P>0.05, OE vs OE + siRNA, P<0.01, 72 hours. D. Cell migration assay for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. E. Colony formation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. F. Representative images of immunohistochemical staining for CLND4 in early-stage glioma tissues (stage I-II, n=15) and late-stage glioma tissues (stage III-IV, n=15); scale bar =100 μm.

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Expressing, Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Over Expression, Cell Migration Assay, Immunohistochemical staining, Staining

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: The CLDN4/NNAT axis modulates glioma progression through Wnt signaling. (A and B) The results of the KEGG enrichment analysis for the major signaling pathways involved in glioma progression in 513 glioma patients divided into CLDN4-high/low (A) or NNAT-high/low (B) groups. (C) Western blotting for Wnt1, Wnt2, and Wnt3A in LN229 cells transfected with empty vector or CLDN4-overexpression vector. (D) Western blotting for Wnt3A in LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with scramble or NNAT siRNAs. (E) Cell proliferation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM). LN229, vector vs vector + PP, P>0.05, OE vs OE + PP, P<0.05, 72 hours; T98G, vector vs vector + PP, P>0.05, OE vs OE + PP, P<0.05, 72 hours. (F) Cell migration assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM). (G) Colony formation assays for LN229/T98G cells transfected with empty vector or CLDN4-overexpression vector treated with PBS or pyrvinium pamoate (10 nM).

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: Protein-Protein interactions, Western Blot, Transfection, Plasmid Preparation, Over Expression, Migration

Journal: American Journal of Cancer Research

Article Title: Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling

doi:

Figure Lengend Snippet: CLDN4 facilitates glioma growth in vivo. A. Western blotting for CLDN4 in primary glioma cells derived from 6 patients. B. Representative images of PDOs derived from CLDN4-high and -low glioma tissues; scale bar =50 μm. C. Western blotting for Wnt3A and NNAT in PDOs derived from CLDN4-high and -low glioma tissues. D. Representative images for immunofluorescence staining for Ki67 in PDOs derived from CLDN4-high and -low glioma tissues; scale bar =50 μm. E. Tumor volumes of subcutaneous tumors formed by LN229 cells transfected with empty vector or CLDN4-overexpression vector (n=5 in each group). F. Kaplan-Meier survival curve for mice with tumors formed by LN229 cells transfected with empty vector or CLDN4-overexpression vector (n=5 in each group).

Article Snippet: Transfected LN229 and T98G cells overexpressing CLDN4 were obtained from GenScript ProBio (China); the same cells transfected with the empty vector (without the CLDN4 gene) were also obtained from the same source.

Techniques: In Vivo, Western Blot, Derivative Assay, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Over Expression

Journal: Scientific Reports

Article Title: A Benzothiazole Derivative (5g) Induces DNA Damage And Potent G2/M Arrest In Cancer Cells

doi: 10.1038/s41598-017-02489-3

Figure Lengend Snippet: Evaluation of antiproliferative activity of 5g in various cancer cells. ( A ) 2-dimensional structure of 5g. ( B ) Antiproliferative activity of 5g (0, 1, 10, 50 and 100 µM at 48 h) was tested in Nalm6, Molt4, CEM, EAC, HCT116, T98G, MCF7 and HeLa cells using MTT assay. ( C ) Table showing observed GI50 values ± SEM of 5g in various cancer cell lines.

Article Snippet: Human cancer cell lines, MCF7 (human breast cancer), K562 (chronic myelogenous leukemia), Molt4 (acute lymphoblastic leukemia), CEM (T-cell leukemia), T98G (human glioblastoma multiforme tumor), HeLa (cervical cancer tumor) and EAC (mouse breast cancer) cells were purchased from National Centre for Cell Science, Pune, India and Nalm6 (B-cell leukemia) and REH (B-cell leukemia) cells were kind gifts from Dr. M.R.

Techniques: Activity Assay, MTT Assay