Journal: Scientific reports

Article Title: PAFAH1B3 is a KLF9 target gene that promotes proliferation and metastasis in pancreatic cancer.

doi: 10.1038/s41598-024-59427-3

Figure Lengend Snippet: Figure 6. PAFAH1B3 affects the expression of EMT-related proteins in pancreatic cancer cells (A) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC, and the protein expression levels of PAFAH1B3, E-cadherin, N-cadherin, Vimentin, Snail1 and MMP2 were measured via Western blotting. (B) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing sh-PAFAH1B3 or sh-NC, and the protein expression levels of PAFAH1B3, E-cadherin, N-cadherin, Vimentin, Snail1 and MMP2 were measured via Western blotting. β-Actin was used as an internal control. The data represent the average of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: rabbit anti-PAFAH1B3 polyclonal antibody (1:1000; Abcam, Cambridge, MA, USA), mouse anti-β-actin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-PCNA polyclonal antibody (1:1000; Abcam, Cambridge, MA, USA), rabbit anti-E-cadherin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-Ncadherin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-Snail1 polyclonal antibody 3 Vol.

Techniques: Expressing, Transduction, Western Blot, Control

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: ( A ) By the immunofluorescence method, the expression of E-cadherin and p120ctn were noted to be restricted to the cell membrane at cell-cell adherens junctions in HBE and H460cells, whereas in SPC and LTE cells, E-cadherin and P120ctn were confined to the cytoplasm. ( B ) Western blot analyses confirmed E-cadherin and p120ctn were expressed in all four cell lines. Of all p120ctn isoforms, only two isoforms, p120ctn isoforms 1 (120 kDa) and 3 (100 kDa), were detected.

Article Snippet: Primary antibodies are as follows: anti-p120ctn mouse monoclonal antibody (1∶200, 610134, BD Transduction Laboratories, USA), anti-E-cadherin rabbit polyclonal antibody (1∶100, SC-7870, Santa Cruz Biotechnology, CA, USA) and anti-E-cadherin mouse monoclonal antibody (1∶100, SC-8426, Santa Cruz Biotechnology, CA, USA).

Techniques: Immunofluorescence, Expressing, Membrane, Western Blot

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: H460 cells were transiently transfected with GFP-siRNA-p120ctn plasmids or with empty vector as control. 24 hours after transfection, an aliquot of the cells was transfected again with p120ctn isoform 1A or 3A cDNA plasmids. ( A ) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal shown in the nucleus and cytoplasm indicates effective expression of GFP from GFP-siRNA-P120ctn, confirming the successful transfection. Depletion of p120ctn (I) reduced the E-cadherin levels (II). Transfection with empty vector (III) did not affect the E-cadherin levels (IV). Restitution of p120ctn isoform 1A (V) restored the E-cadherin on the cell membrane (VI), while restitution of p120ctn isoform 3A (VII) had no effects on E-cadherin levels (VIII). ( B ) Levels of E-cadherin were then analyzed by Western blot assay. The results confirmed that depletion of p120ctn resulted in decreased E-cadherin levels; restitution of p120ctn isoform 1A restored the E-cadherin levels, while restitution of p120ctn isoform 3A had no effects on E-cadherin expression. ( C ) The invasiveness of H460 cells were analyzed by Matrigel invasion assay. P120ctn ablation enhanced the invasiveness in comparison with the control group transfected with vector alone (*, P <0.01). Restitution of p120ctn isoform 1A reduced the invasiveness of H460 cells in comparison with the group with p120ctn ablation (Si-p120ctn) (**, P <0.01), while p120ctn isoform 3A increased the invasiveness (***, P <0.01).

Article Snippet: Primary antibodies are as follows: anti-p120ctn mouse monoclonal antibody (1∶200, 610134, BD Transduction Laboratories, USA), anti-E-cadherin rabbit polyclonal antibody (1∶100, SC-7870, Santa Cruz Biotechnology, CA, USA) and anti-E-cadherin mouse monoclonal antibody (1∶100, SC-8426, Santa Cruz Biotechnology, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Expressing, Membrane, Western Blot, Invasion Assay, Comparison

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: SPC cells were transiently transfected with GFP-siRNA-p120ctn or with empty vector as control. 24 hours after transfection, an aliquot of cells was transfected again with p120ctn isoform 1A or 3A cDNA plasmids. ( A ) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal shown in the nucleus and cytoplasm indicates effective expression of GFP from GFP-siRNA-P120ctn, confirming the successful transfection. Depletion of p120ctn (I) reduced the E-cadherin levels (II). Transfection with empty vector (III) did not affect the E-cadherin levels (IV). Restitution of p120ctn isoform 1A (V) restored the cytoplasmic E-cadherin levels (VI), while restitution of p120ctn isoform 3A (VII) had no effects on the E-cadherin levels (VIII). ( B ) Levels of E-cadherin were then analyzed by Western blot assay. The results confirmed that depletion of p120ctn resulted in decreased E-cadherin levels. Restitution of p120ctn isoform 1A restored the E-cadherin levels, while restitution of p120ctn isoform 3A had no effects on E-cadherin expression. ( C ) The invasiveness of SPC cells were analyzed by Matrigel invasion assay. P120ctn ablation reduced the cell invasiveness in comparison with the control group transfected with vector alone (*, P <0.01). Restitution of p120ctn isoform 1A and 3A both enhanced the invasiveness of SPC cells in comparison with the group with p120ctn ablation (Si-p120ctn) (**, P <0.01, ***, P <0.01).

Article Snippet: Primary antibodies are as follows: anti-p120ctn mouse monoclonal antibody (1∶200, 610134, BD Transduction Laboratories, USA), anti-E-cadherin rabbit polyclonal antibody (1∶100, SC-7870, Santa Cruz Biotechnology, CA, USA) and anti-E-cadherin mouse monoclonal antibody (1∶100, SC-8426, Santa Cruz Biotechnology, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Expressing, Western Blot, Invasion Assay, Comparison

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: H460 cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). ( A ) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn, M1–M3, and M5-GFP was expressed in the nucleus and cytoplasm. GFP from GFP-si-P120ctn and M4-GFP was expressed in the nucleus, cytoplasm and on the cell membrane (IX). Expression or repletion of M4 mutant (IX) restored E-cadherin on the cell membrane (X), while expression of the other mutants had no significant effects on E-cadherin levels. ( B ) The levels of E-cadherin were then analyzed by Western blot assay. Expression of M1–M5 mutants were detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated the E-cadherin levels, whereas the other mutants had no effects on the E-cadherin levels. ( C ) The invasiveness of H460 cells was analyzed by Matrigel invasion assay. Repletion of M4 mutant reduced the cell invasiveness in comparison with the group of p120ctn ablation (Si-p120ctn) (**, P <0.01), while repletion of M2 mutant enhanced the invasiveness (*, P <0.01). Repletion of the other mutants (M1, M3 and M5) did not show significant effects on cell invasiveness in comparison with the group of 120ctn ablation.

Article Snippet: Primary antibodies are as follows: anti-p120ctn mouse monoclonal antibody (1∶200, 610134, BD Transduction Laboratories, USA), anti-E-cadherin rabbit polyclonal antibody (1∶100, SC-7870, Santa Cruz Biotechnology, CA, USA) and anti-E-cadherin mouse monoclonal antibody (1∶100, SC-8426, Santa Cruz Biotechnology, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Expressing, Construct, Membrane, Mutagenesis, Western Blot, Invasion Assay, Comparison

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: SPC cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). ( A ) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn or M1–M5-GFP was expressed in the nucleus and cytoplasm. Expression (repletion) of M4 mutant (IX) restored cytoplasmic E-cadherin (X), while expression of the other mutants had no significant effects on the E-cadherin levels. ( B ) The levels of E-cadherin were analyzed by Western blot assay. Expression of M1–M5 mutants was detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated E-cadherin levels, whereas expression of the other mutants had no effects on the E-cadherin levels. ( C ) The invasiveness of SPC cells was analyzed by Matrigel invasion assay. Expression of M2 or M4 mutants enhanced the cell invasiveness in comparison with the group with ablated p120ctn (Si-p120ctn) (*, P <0.01, **, P <0.01), while expression of other three p120 1A mutants did not show significant effects.

Article Snippet: Primary antibodies are as follows: anti-p120ctn mouse monoclonal antibody (1∶200, 610134, BD Transduction Laboratories, USA), anti-E-cadherin rabbit polyclonal antibody (1∶100, SC-7870, Santa Cruz Biotechnology, CA, USA) and anti-E-cadherin mouse monoclonal antibody (1∶100, SC-8426, Santa Cruz Biotechnology, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Expressing, Construct, Mutagenesis, Western Blot, Invasion Assay, Comparison