Journal: International Journal of Biological Sciences

Article Title: KLF4-Mediated CDH3 Upregulation Suppresses Human Hepatoma Cell Growth and Migration via GSK-3β Signaling

doi: 10.7150/ijbs.30857

Figure Lengend Snippet: Direct transcriptional activation of CDH3 by KLF4 in HCC cells. (A) The two potential binding sites for KLF4 in the promoter of CDH3. (B) CDH3 promoter reporters (pGL3-CDH3-627 and pGL3-CDH3-439) were transfected into 239T cells in triplicate with KLF4 expression plasmids or control vectors for 24 h. The CDH3 promoter activity was then examined using a dual luciferase assay kit. (C) The CDH3 promoter reporter activities were determined in KLF4 overexpressed Huh7 and Hep3B cells. (D) The CDH3 promoter reporter activities were determined in KLF4 silenced YY-8103 and HCC-LM3 cells. The experiments were performed independently three times. *P<0.05, **P<0.01. (E) Chromatin immunoprecipitation assays were performed with a specific anti-KLF4 antibody or IgG and oligonucleotides flanking the KLF4 binding sites were amplified by PCR. ( F ) The activities of mutant CDH3 promoters were determined by dual luciferase assays in KLF4 overexpressed Huh7 cells.

Article Snippet: Immunohistochemical analysis was performed with anti-KLF4 (1:200, Proteintech, 11880-1-AP) and anti-CDH3 antibody (1:50, Proteintech, 13773-1-AP).

Techniques: Activation Assay, Binding Assay, Transfection, Expressing, Control, Activity Assay, Luciferase, Chromatin Immunoprecipitation, Amplification, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: KLF4-Mediated CDH3 Upregulation Suppresses Human Hepatoma Cell Growth and Migration via GSK-3β Signaling

doi: 10.7150/ijbs.30857

Figure Lengend Snippet: CDH3 expression is activated by KLF4 in HCC cells. (A) KLF4 overexpression by transient transfection promoted CDH3 expression by qRT-PCR. (B) The mRNA level of CDH3 detected by qRT-PCR was reduced in KLF4 stably silenced HCC cells. *P<0.05, **P<0.01. (C) Overexpression of KFL4 enhanced CDH3 expression at protein level in WRL68 and YY-8103 cells detected by western blot analyses. (D) Western blots showing the expression pattern of KLF4 and CDH3 in a series of HCC cell lines.

Article Snippet: Immunohistochemical analysis was performed with anti-KLF4 (1:200, Proteintech, 11880-1-AP) and anti-CDH3 antibody (1:50, Proteintech, 13773-1-AP).

Techniques: Expressing, Over Expression, Transfection, Quantitative RT-PCR, Stable Transfection, Western Blot

Journal: International Journal of Biological Sciences

Article Title: KLF4-Mediated CDH3 Upregulation Suppresses Human Hepatoma Cell Growth and Migration via GSK-3β Signaling

doi: 10.7150/ijbs.30857

Figure Lengend Snippet: CDH3 expression is associated with KLF4 level in HCC specimens. (A) TMA specimens including 64 HCC tissues were prepared for immunostaining with specific antibodies against CDH3 and KLF4. Representative images of CDH3 and KLF4 expression in HCC specimens were shown. (B) Data showed that the CDH3 expression levels directly correlated with the KLF4 expression levels in HCC. (C) A positive correlation between KLF4 and CDH3 mRNA level in additional 37 paired HCC tissues was measured by linear regression (r=0.675, P<0.001).

Article Snippet: Immunohistochemical analysis was performed with anti-KLF4 (1:200, Proteintech, 11880-1-AP) and anti-CDH3 antibody (1:50, Proteintech, 13773-1-AP).

Techniques: Expressing, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: KLF4-Mediated CDH3 Upregulation Suppresses Human Hepatoma Cell Growth and Migration via GSK-3β Signaling

doi: 10.7150/ijbs.30857

Figure Lengend Snippet: Downregulation of CDH3 induced cell proliferation and migration. (A) Colony formation assays were performed to detect the growth of LV-shCDH3-infected Focus, HCC-LM3 and WRL68 cells and control cells, respectively. Colonies were counted and captured. The levels of CDH3 expression were examined via western blot. *P<0.05. (B) Transwell assays were performed to detect changes in migratory abilities of Focus, HCC-LM3 and WRL68 cells treated with LV-shCDH3 or LV-shNC control. (C) KLF4 expression plasmid was transfected into CDH3 silenced HCC cells and control cells, respectively. Transwell chamber assays were then performed to detect cell migration of these cells.

Article Snippet: Immunohistochemical analysis was performed with anti-KLF4 (1:200, Proteintech, 11880-1-AP) and anti-CDH3 antibody (1:50, Proteintech, 13773-1-AP).

Techniques: Migration, Infection, Control, Expressing, Western Blot, Plasmid Preparation, Transfection

Journal: International Journal of Biological Sciences

Article Title: KLF4-Mediated CDH3 Upregulation Suppresses Human Hepatoma Cell Growth and Migration via GSK-3β Signaling

doi: 10.7150/ijbs.30857

Figure Lengend Snippet: The effect of CDH3 overexpression on growth and migration of HCC cells. (A) Cell proliferation of Huh7, YY-8103 and Sk-Hep-1 cells transfected with CDH3 plasmid or control empty vector was evaluated using CCK8 assay. And the level of CDH3 expression was examined using western blot. *P<0.05. (B) Transwell assays were conducted to evaluate migration of CDH3 overexpressed Huh7, YY-8103 and Sk-Hep-1 cells.

Article Snippet: Immunohistochemical analysis was performed with anti-KLF4 (1:200, Proteintech, 11880-1-AP) and anti-CDH3 antibody (1:50, Proteintech, 13773-1-AP).

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Control, CCK-8 Assay, Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: KLF4-Mediated CDH3 Upregulation Suppresses Human Hepatoma Cell Growth and Migration via GSK-3β Signaling

doi: 10.7150/ijbs.30857

Figure Lengend Snippet: CDH3 affects GSK-3β expression in HCC cells. (A) Western blot analysis was used to measure the expressions of GSK-3β, p-GSK-3β, AKT, p-AKT, Erk, p-Erk, P70S6K, p-P70S6K, SMAD3, p-SMAD and β-catenin in CDH3 silenced Focus cells. (B) The levels of GSK-3β and p-GSK-3β were examined in CDH3 overexpressed YY8103 and HCC-LM3 cells using western blot assay. The cells were treated with or without insulin for 10 min. (C) TCF/LEF luciferase activity was measured by dual luciferase reporter kit and the results illustrated that CDH3 expression had no effect on the transcription activity of Wnt/β-catenin signaling. (D) The GSK-3β knockdown cell line was constructed by transfecting siGSK-3β into Focus and YY-8103 cells and detected by western blotting. (E) The migration assay was conducted in Focus and YY-8103 cells after transfection with siGSK-3β (or control).

Article Snippet: Immunohistochemical analysis was performed with anti-KLF4 (1:200, Proteintech, 11880-1-AP) and anti-CDH3 antibody (1:50, Proteintech, 13773-1-AP).

Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Knockdown, Construct, Migration, Transfection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members

doi: 10.1073/pnas.1809430115

Figure Lengend Snippet: Cadherin expression in Cdh3-GFP-RGCs. (A) We propose that DSCAM masks inappropriate adhesion, allowing indifference between homotypic neurites. Without DSCAM, unmasked adhesion drives clustering and fasciculation, predicting that reducing adhesion will partially rescue fasciculation, and that ectopic overexpressing of a CAM will make random cells fasciculate together as if they were homotypic. (B–E) In situ hybridization (RNAscope), with probes against GFP (green), Cdh3 (red), and Cdh6 (cyan), performed on P0 Cdh3-GFP retinas. Forty-eight of 50 GFP-positive cells were positive for both Cdh3 and Cdh6 (dotted lines). Many GFP-negative cells were Cdh6 positive (filled arrowheads in B and C), while occasional cells that were GFP-negative but Cdh3/Cdh6 double positive were also observed (open arrowheads in D and E, n = 6 retinas). (Scale bar: 50 μm.)

Article Snippet: An expression construct containing Myc-DDK–tagged cadherin-3 (pLenti-Cdh3) was obtained from Origene (MR227582L1).

Techniques: Expressing, In Situ Hybridization

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members

doi: 10.1073/pnas.1809430115

Figure Lengend Snippet: Cadherin-mediated adhesion contributes to Cdh3-GFP-RGC fasciculation. Confocal image projections through Cdh3-GFP-RGC dendrites in whole-mount retinas from wild-type (A), Cdh3−/− (B), Cdh6−/− (C), and Ctnna2CDF/+ (D) mice and from all genotypes in combination with Dscam−/− (E–H) demonstrate that dendrite fascicles are less severe in double mutants than in Dscam−/− alone (arrowheads). Images were quantified using the FS (I) and the Elo score (J). n = 4 to 12 retinas per genotype (actual n values are noted in J) over one to four microscope fields of view (median, 3 fields per retina). Box plots represent the median, first and third quartiles, range, and outliers. Blue diamonds in I represent the means. *P < 0.05, **P < 0.01, and ***P < 0.001 by pairwise Wilcoxon rank sum test compared with Dscam−/−. (Scale bar: 100 μm.)

Article Snippet: An expression construct containing Myc-DDK–tagged cadherin-3 (pLenti-Cdh3) was obtained from Origene (MR227582L1).

Techniques: Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members

doi: 10.1073/pnas.1809430115

Figure Lengend Snippet: Cdh3 overexpression can drive fasciculation between nonhomotypic cells. Retinas of Vgat-Cre mice were electroporated in vivo at P0 with the Cre-inducible expression construct pCALNL-DsRed, alone or along with pCALNL-Cdh3. After 2 wk, these were fixed, stained for DsRed, and imaged en face. (A and B) Mice wild-type for Dscam (A) and DscamcKO/cKO mutants with conditional mutation in GABAergic amacrine cells (B) were electroporated with DsRed alone (Upper). There was no appreciable fasciculation (depicted in Lower) between electroporated cells. (C) Likewise, when retinas wild-type for Dscam were coelectroporated with DsRed and Cdh3 (Upper), no fasciculation (depicted in Lower) was observed. (D) In contrast, when DsRed and Cdh3 were together introduced into DscamcKO/cKO mutant retinas (Upper), significant fasciculation (depicted in Lower) was observed (arrowheads). (E) Images were compared for fasciculation using the Elo score. P = 0.025 by pairwise Wilcoxon rank sum test compared with each other condition. n = 6 mice per condition. (Scale bar: 100 μm.)

Article Snippet: An expression construct containing Myc-DDK–tagged cadherin-3 (pLenti-Cdh3) was obtained from Origene (MR227582L1).

Techniques: Over Expression, In Vivo, Expressing, Construct, Staining, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members

doi: 10.1073/pnas.1809430115

Figure Lengend Snippet: Trans DSCAM interactions mask the CDH3 adhesive response. Cortical neurons from wild-type (A and D) and Dscam−/− (B and E) mice were transfected with constructs encoding CDH3 with a C-terminal FLAG tag (green) and then incubated for 1 h with beads coated with CDH3 and DSCAM ectodomains (A and B, magenta) or CDH3-EC alone (D and E, magenta). The accumulation of CDH3-FLAG at sites of contact between beads and neurons was quantified (C and F). In all four conditions, CDH3 was present both in the neuron and on the bead. While neuronal CDH3-FLAG was largely indifferent to the beads when DSCAM was both in the neuron and on the bead (A), CDH3 accumulated at these sites when DSCAM was present only on the bead (B), only in the neuron (D), or completely absent (E), demonstrating that trans DSCAM interactions masked this accumulation. Means ± SEM are presented in C and F. n = 20 to 31 neurons per condition, from cultures separately prepared from three different mice per genotype (actual n values are noted in C and F). ***P < 0.001 by two-tailed Student’s t test. (Scale bar: 10 μm.)

Article Snippet: An expression construct containing Myc-DDK–tagged cadherin-3 (pLenti-Cdh3) was obtained from Origene (MR227582L1).

Techniques: Transfection, Construct, FLAG-tag, Incubation, Two Tailed Test