plac8  (OriGene)

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 1. Induction of PLAC8 leads to defective autophagy and transformation in prostate epithelial cells exposed to Cd. (A) Western blot analysis confirming induc- tion of autophagy signaling following chronic exposure to Cd in prostate epithelial cells. (B) Immunofluorescence of RWPE-1 and Cd-transforming cells shows an increase in percentage of cells with LC3B and PLAC8 fusion. (C) Immunofluorescence of RWPE-1 and Cd-transforming cells shows a decrease in percentage of cells with LC3B and LAMP-1fusion. (D) Immunofluorescence staining and the colocalization analysis of LC3B with LAMP1 and PLAC8 were assessed using Pearson coefficient. (E) Representa- tive TEM images illustrating the fusion of autophagosomes and lysosomes in RWPE-1 and CTPE cells, along with quantification of autophagosomes, lysosomes, and au- tolysosomes per square micrometer. (F) The expression levels of PLAC8, LAMP1, and LC3B were determined by Western blot analysis in shRNA-PLAC8–transfected cells, both in the presence and absence of Cd. Veh, vehicle. (G) Immunofluorescence staining and colocalization analysis of LC3B and LAMP1 fusion with increased Pearson coefficient in sh-PLAC8 CTPE cells. (H) Representative TEM images showing fusion of autophagosomes and lysosomes in shRNA PLAC8-transfected CTPE cells compared to vector alone, along with the quantification of autophagosomes, lysosomes, and autolysosomes per square micrometer. Arrowheads indicate the following: lysosomes (blue), autophagic vacuoles (red), and autolysosomes (green). All error bars represent means ± SD. Statistical significance: *P < 0.05; ns, not significant.

Article Snippet: Cd chloride hydrate (0.1 M) was purchased from Hampton Research (USA), CHX was purchased from Sigma- Aldrich (St. Louis, USA), BD Matrigel was purchased from Corning (NY, USA), A- 1331852 (BCLxL inhibitor) was purchased from MedChemExpress (NJ, USA), and LPS was purchased from Sigma- Aldrich (St. Louis, USA). shRNA/small interfering RNA/pCMV transfections Cd- transformed RWPE- 1 cells with stable PLAC8 (TL302451, Origene), NF- κB (TL302038, Origene), or BCL- xL (TR320077, Origene) knockdown were generated by shRNA and antibiotic selection.

Techniques: Transformation Assay, Western Blot, Immunofluorescence, Staining, Expressing, shRNA, Transfection, Plasmid Preparation

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 2. Knocking down PLAC8 expression inhibits Cd-induced tumor growth in xenotransplanted mice. (A) In CTPE cells, silencing PLAC8 expression reduced tumor formation in the xenotransplantation model. (B) Immunohistochemistry (IHC) of tumor tissues analyzed for Ki-67, PLAC8, LC3b, and LAMP1 expression. (C) A volcano plot analysis displayed the differential expression of genes in sh-PLAC8 tumors compared to the control group. (D) GSEA identified pathways associated with prostate cancer, lysosomal functions, and NF-κB–mediated TNF-α signaling in PLAC8-knockdown (PLAC8_KD) tumors compared to the vector control. (E) Cd-transforming cells showed a time-dependent induction of p65 expression (F) and NF-κB activation was observed. (G) Both cytosolic and nuclear expression of p65 were noted during the transforma- tion of Cd-exposed RWPE-1 cells. (H) p65 binding sites on the PLAC8 promoter were identified and validated by comparing luciferase activity in wild-type and mutated (Δ) sites, transcription start sites (TSS) and (I) ChIP-qPCR was performed in CTPE cells. All error bars represent means ± SD, with statistical significance indicated as *P < 0.05, ***P < 0.001; ns, not significant. NES, normalized enrichment score.

Article Snippet: Cd chloride hydrate (0.1 M) was purchased from Hampton Research (USA), CHX was purchased from Sigma- Aldrich (St. Louis, USA), BD Matrigel was purchased from Corning (NY, USA), A- 1331852 (BCLxL inhibitor) was purchased from MedChemExpress (NJ, USA), and LPS was purchased from Sigma- Aldrich (St. Louis, USA). shRNA/small interfering RNA/pCMV transfections Cd- transformed RWPE- 1 cells with stable PLAC8 (TL302451, Origene), NF- κB (TL302038, Origene), or BCL- xL (TR320077, Origene) knockdown were generated by shRNA and antibiotic selection.

Techniques: Expressing, Immunohistochemistry, Quantitative Proteomics, Control, Knockdown, Plasmid Preparation, Activation Assay, Binding Assay, Luciferase, Activity Assay, ChIP-qPCR

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 3. The interaction between PLAC8 and NF-κB during the transformation of prostate epithelial cells. (A) The interaction between p65 and PLAC8 is confirmed by immunoprecipitation (IP) analysis. IgG, immunoglobulin G. (B) CHX was used to inhibit protein synthesis in vector alone and sh-p65 cells, and Western blot (WB) analysis was performed to show that p65 is necessary to stabilize PLAC8 in CTPE cells. h, hours. (C) Immunofluorescence of RWPE-1 and Cd-transforming cells shows an increase in percent- age of cells with PLAC8 and p65 colocalization. (D) Immunofluorescence staining and the colocalization analysis of p65 and PLAC8 were assessed using Pearson coefficient. (E) Ectopic expression of p65 increases PLAC8 expression in RWPE-1 cells. (F) The expression levels of p65, PLAC8, LAMP1, and LC3B were determined by Western blot analysis in sh-p65–transfected cells, both in the presence and absence of Cd. (G) Immunofluorescence staining and colocalization analysis of LC3B and LAMP1 fusion with increased Pearson coefficient in sh-p65 CTPE cells. (H) Representative TEM images showing fusion of autophagosomes and lysosomes in sh-p65–transfected CTPE cells compared to vector alone, along with the quantification of autophagosomes, lysosomes, and autolysosomes per square micrometer. Arrowheads indicate lysosomes (in blue), autophagic vacuoles (in red), and autolysosomes (in green). All error bars represent means ± SD. Statistical significance is indicated as *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: Cd chloride hydrate (0.1 M) was purchased from Hampton Research (USA), CHX was purchased from Sigma- Aldrich (St. Louis, USA), BD Matrigel was purchased from Corning (NY, USA), A- 1331852 (BCLxL inhibitor) was purchased from MedChemExpress (NJ, USA), and LPS was purchased from Sigma- Aldrich (St. Louis, USA). shRNA/small interfering RNA/pCMV transfections Cd- transformed RWPE- 1 cells with stable PLAC8 (TL302451, Origene), NF- κB (TL302038, Origene), or BCL- xL (TR320077, Origene) knockdown were generated by shRNA and antibiotic selection.

Techniques: Transformation Assay, Immunoprecipitation, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Expressing, Transfection

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 4. Knockdown of p65 inhibits Cd-induced tumor growth in xenotransplanted mice. (A) Confirmation of stable p65 knockdown in CTPE cells via Western blot analysis (left side), with selected clones inoculated into nude mice to assess tumor inhibition. (B) A volcano plot analysis illustrates the differential expression of genes in sh-p65 tumors compared to the vehicle group. (C) GSEA plot shows pathways involved in proteasome degradation, autophagy, and apoptosis in sh-p65 tumors compared to the vector control. (D) IHC analysis was performed to determine the expressions of Ki-67, p65, PLAC8, LC3B, and LAMP1 in xenograft tumors from the vector and sh-p65 groups. (E) Protein expression levels of p65, PLAC8, LC3B, and LAMP1 in xenograft tumors from sh-p65 and vector-only groups. All error bars represent means ± SD, with ***P < 0.001.

Article Snippet: Cd chloride hydrate (0.1 M) was purchased from Hampton Research (USA), CHX was purchased from Sigma- Aldrich (St. Louis, USA), BD Matrigel was purchased from Corning (NY, USA), A- 1331852 (BCLxL inhibitor) was purchased from MedChemExpress (NJ, USA), and LPS was purchased from Sigma- Aldrich (St. Louis, USA). shRNA/small interfering RNA/pCMV transfections Cd- transformed RWPE- 1 cells with stable PLAC8 (TL302451, Origene), NF- κB (TL302038, Origene), or BCL- xL (TR320077, Origene) knockdown were generated by shRNA and antibiotic selection.

Techniques: Knockdown, Western Blot, Clone Assay, Inhibition, Quantitative Proteomics, Plasmid Preparation, Control, Expressing

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 5. BCL-xL plays a crucial role in the survival of transformed cells and is regulated by PLAC8. (A) Inhibiting the expression of p65 and PLAC8 enhances the induc- tion of apoptosis in CTPE cells, confirmed by flow cytometry analysis of annexin V-FITC–stained apoptotic cells. (B) Ectopic expression of PLAC8 leads to increased levels of BCL-xL and p65 in RWPE-1 cells. (C) The expression of BCL-xL is observed at successive stages of Cd exposure during the transformation of RWPE-1 cells. (D) Silencing BCL-xL expression abolishes the PLAC8-mediated autophagy signaling in CTPE cells. (E) Ectopic expression of BCL-xL results in up-regulating PLAC8 and p65 in RWPE-1 cells. (F) In CTPE cells, cotransfection with sh-PLAC8 and the pCMV p65 overexpression plasmid demonstrated PLAC8, p65, and BCL-xL protein levels through Western blot analysis. (G) A luciferase assay showing increased BCL-xL promoter activity in CTPE cells compared to RWPE-1 cells. All error bars represent means ± SD, with statistical significance at *P < 0.05.

Article Snippet: Cd chloride hydrate (0.1 M) was purchased from Hampton Research (USA), CHX was purchased from Sigma- Aldrich (St. Louis, USA), BD Matrigel was purchased from Corning (NY, USA), A- 1331852 (BCLxL inhibitor) was purchased from MedChemExpress (NJ, USA), and LPS was purchased from Sigma- Aldrich (St. Louis, USA). shRNA/small interfering RNA/pCMV transfections Cd- transformed RWPE- 1 cells with stable PLAC8 (TL302451, Origene), NF- κB (TL302038, Origene), or BCL- xL (TR320077, Origene) knockdown were generated by shRNA and antibiotic selection.

Techniques: Transformation Assay, Expressing, Flow Cytometry, Staining, Cotransfection, Over Expression, Plasmid Preparation, Western Blot, Luciferase, Activity Assay

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 6. Inhibition of BCL-xL suppresses PLAC8-mediated tumorigenesis in a xenotransplanted model. (A) The intraperitoneal injection of a pharmacological inhibi- tor of BCL-xL (A-1155643) and (B) stably suppressing BCL-xL in CTPE cells significantly inhibits tumor growth. (C) IHC analysis of Ki-67, p65, PLAC8, LC3B, and LAMP1 ex- pression in both vector and sh–BCL-xL groups. (D) A volcano plot analysis demonstrated the differential expression of genes in the shBCL-xL tumors compared to the vehicle group. (E) GSEA revealed alterations in the unfolded protein response, autophagy, and apoptosis pathways in sh–BCL-xL tumors compared to the vector group. All error bars represent means ± SD. **P < 0.01 and ****P < 0.0001.

Article Snippet: Cd chloride hydrate (0.1 M) was purchased from Hampton Research (USA), CHX was purchased from Sigma- Aldrich (St. Louis, USA), BD Matrigel was purchased from Corning (NY, USA), A- 1331852 (BCLxL inhibitor) was purchased from MedChemExpress (NJ, USA), and LPS was purchased from Sigma- Aldrich (St. Louis, USA). shRNA/small interfering RNA/pCMV transfections Cd- transformed RWPE- 1 cells with stable PLAC8 (TL302451, Origene), NF- κB (TL302038, Origene), or BCL- xL (TR320077, Origene) knockdown were generated by shRNA and antibiotic selection.

Techniques: Inhibition, Injection, Stable Transfection, Plasmid Preparation, Quantitative Proteomics

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 1. Induction of PLAC8 leads to defective autophagy and transformation in prostate epithelial cells exposed to Cd. (A) Western blot analysis confirming induc- tion of autophagy signaling following chronic exposure to Cd in prostate epithelial cells. (B) Immunofluorescence of RWPE-1 and Cd-transforming cells shows an increase in percentage of cells with LC3B and PLAC8 fusion. (C) Immunofluorescence of RWPE-1 and Cd-transforming cells shows a decrease in percentage of cells with LC3B and LAMP-1fusion. (D) Immunofluorescence staining and the colocalization analysis of LC3B with LAMP1 and PLAC8 were assessed using Pearson coefficient. (E) Representa- tive TEM images illustrating the fusion of autophagosomes and lysosomes in RWPE-1 and CTPE cells, along with quantification of autophagosomes, lysosomes, and au- tolysosomes per square micrometer. (F) The expression levels of PLAC8, LAMP1, and LC3B were determined by Western blot analysis in shRNA-PLAC8–transfected cells, both in the presence and absence of Cd. Veh, vehicle. (G) Immunofluorescence staining and colocalization analysis of LC3B and LAMP1 fusion with increased Pearson coefficient in sh-PLAC8 CTPE cells. (H) Representative TEM images showing fusion of autophagosomes and lysosomes in shRNA PLAC8-transfected CTPE cells compared to vector alone, along with the quantification of autophagosomes, lysosomes, and autolysosomes per square micrometer. Arrowheads indicate the following: lysosomes (blue), autophagic vacuoles (red), and autolysosomes (green). All error bars represent means ± SD. Statistical significance: *P < 0.05; ns, not significant.

Article Snippet: To colocate proteins within individual cells, we used conjugated antibodies for LC3B (ab225383 and ab225382), LAMP1 (ab302684), and NF- κB (ab190589 and ab214846) from Abcam, USA and a PLAC8 antibody (CSB- CSB- PA873705LC01HU) from CUSABIO, USA.

Techniques: Transformation Assay, Western Blot, Immunofluorescence, Staining, Expressing, shRNA, Transfection, Plasmid Preparation

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 2. Knocking down PLAC8 expression inhibits Cd-induced tumor growth in xenotransplanted mice. (A) In CTPE cells, silencing PLAC8 expression reduced tumor formation in the xenotransplantation model. (B) Immunohistochemistry (IHC) of tumor tissues analyzed for Ki-67, PLAC8, LC3b, and LAMP1 expression. (C) A volcano plot analysis displayed the differential expression of genes in sh-PLAC8 tumors compared to the control group. (D) GSEA identified pathways associated with prostate cancer, lysosomal functions, and NF-κB–mediated TNF-α signaling in PLAC8-knockdown (PLAC8_KD) tumors compared to the vector control. (E) Cd-transforming cells showed a time-dependent induction of p65 expression (F) and NF-κB activation was observed. (G) Both cytosolic and nuclear expression of p65 were noted during the transforma- tion of Cd-exposed RWPE-1 cells. (H) p65 binding sites on the PLAC8 promoter were identified and validated by comparing luciferase activity in wild-type and mutated (Δ) sites, transcription start sites (TSS) and (I) ChIP-qPCR was performed in CTPE cells. All error bars represent means ± SD, with statistical significance indicated as *P < 0.05, ***P < 0.001; ns, not significant. NES, normalized enrichment score.

Article Snippet: To colocate proteins within individual cells, we used conjugated antibodies for LC3B (ab225383 and ab225382), LAMP1 (ab302684), and NF- κB (ab190589 and ab214846) from Abcam, USA and a PLAC8 antibody (CSB- CSB- PA873705LC01HU) from CUSABIO, USA.

Techniques: Expressing, Immunohistochemistry, Quantitative Proteomics, Control, Knockdown, Plasmid Preparation, Activation Assay, Binding Assay, Luciferase, Activity Assay, ChIP-qPCR

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 3. The interaction between PLAC8 and NF-κB during the transformation of prostate epithelial cells. (A) The interaction between p65 and PLAC8 is confirmed by immunoprecipitation (IP) analysis. IgG, immunoglobulin G. (B) CHX was used to inhibit protein synthesis in vector alone and sh-p65 cells, and Western blot (WB) analysis was performed to show that p65 is necessary to stabilize PLAC8 in CTPE cells. h, hours. (C) Immunofluorescence of RWPE-1 and Cd-transforming cells shows an increase in percent- age of cells with PLAC8 and p65 colocalization. (D) Immunofluorescence staining and the colocalization analysis of p65 and PLAC8 were assessed using Pearson coefficient. (E) Ectopic expression of p65 increases PLAC8 expression in RWPE-1 cells. (F) The expression levels of p65, PLAC8, LAMP1, and LC3B were determined by Western blot analysis in sh-p65–transfected cells, both in the presence and absence of Cd. (G) Immunofluorescence staining and colocalization analysis of LC3B and LAMP1 fusion with increased Pearson coefficient in sh-p65 CTPE cells. (H) Representative TEM images showing fusion of autophagosomes and lysosomes in sh-p65–transfected CTPE cells compared to vector alone, along with the quantification of autophagosomes, lysosomes, and autolysosomes per square micrometer. Arrowheads indicate lysosomes (in blue), autophagic vacuoles (in red), and autolysosomes (in green). All error bars represent means ± SD. Statistical significance is indicated as *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: To colocate proteins within individual cells, we used conjugated antibodies for LC3B (ab225383 and ab225382), LAMP1 (ab302684), and NF- κB (ab190589 and ab214846) from Abcam, USA and a PLAC8 antibody (CSB- CSB- PA873705LC01HU) from CUSABIO, USA.

Techniques: Transformation Assay, Immunoprecipitation, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Expressing, Transfection

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 4. Knockdown of p65 inhibits Cd-induced tumor growth in xenotransplanted mice. (A) Confirmation of stable p65 knockdown in CTPE cells via Western blot analysis (left side), with selected clones inoculated into nude mice to assess tumor inhibition. (B) A volcano plot analysis illustrates the differential expression of genes in sh-p65 tumors compared to the vehicle group. (C) GSEA plot shows pathways involved in proteasome degradation, autophagy, and apoptosis in sh-p65 tumors compared to the vector control. (D) IHC analysis was performed to determine the expressions of Ki-67, p65, PLAC8, LC3B, and LAMP1 in xenograft tumors from the vector and sh-p65 groups. (E) Protein expression levels of p65, PLAC8, LC3B, and LAMP1 in xenograft tumors from sh-p65 and vector-only groups. All error bars represent means ± SD, with ***P < 0.001.

Article Snippet: To colocate proteins within individual cells, we used conjugated antibodies for LC3B (ab225383 and ab225382), LAMP1 (ab302684), and NF- κB (ab190589 and ab214846) from Abcam, USA and a PLAC8 antibody (CSB- CSB- PA873705LC01HU) from CUSABIO, USA.

Techniques: Knockdown, Western Blot, Clone Assay, Inhibition, Quantitative Proteomics, Plasmid Preparation, Control, Expressing

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 5. BCL-xL plays a crucial role in the survival of transformed cells and is regulated by PLAC8. (A) Inhibiting the expression of p65 and PLAC8 enhances the induc- tion of apoptosis in CTPE cells, confirmed by flow cytometry analysis of annexin V-FITC–stained apoptotic cells. (B) Ectopic expression of PLAC8 leads to increased levels of BCL-xL and p65 in RWPE-1 cells. (C) The expression of BCL-xL is observed at successive stages of Cd exposure during the transformation of RWPE-1 cells. (D) Silencing BCL-xL expression abolishes the PLAC8-mediated autophagy signaling in CTPE cells. (E) Ectopic expression of BCL-xL results in up-regulating PLAC8 and p65 in RWPE-1 cells. (F) In CTPE cells, cotransfection with sh-PLAC8 and the pCMV p65 overexpression plasmid demonstrated PLAC8, p65, and BCL-xL protein levels through Western blot analysis. (G) A luciferase assay showing increased BCL-xL promoter activity in CTPE cells compared to RWPE-1 cells. All error bars represent means ± SD, with statistical significance at *P < 0.05.

Article Snippet: To colocate proteins within individual cells, we used conjugated antibodies for LC3B (ab225383 and ab225382), LAMP1 (ab302684), and NF- κB (ab190589 and ab214846) from Abcam, USA and a PLAC8 antibody (CSB- CSB- PA873705LC01HU) from CUSABIO, USA.

Techniques: Transformation Assay, Expressing, Flow Cytometry, Staining, Cotransfection, Over Expression, Plasmid Preparation, Western Blot, Luciferase, Activity Assay

Journal: Science advances

Article Title: Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium.

doi: 10.1126/sciadv.adv8640

Figure Lengend Snippet: Fig. 6. Inhibition of BCL-xL suppresses PLAC8-mediated tumorigenesis in a xenotransplanted model. (A) The intraperitoneal injection of a pharmacological inhibi- tor of BCL-xL (A-1155643) and (B) stably suppressing BCL-xL in CTPE cells significantly inhibits tumor growth. (C) IHC analysis of Ki-67, p65, PLAC8, LC3B, and LAMP1 ex- pression in both vector and sh–BCL-xL groups. (D) A volcano plot analysis demonstrated the differential expression of genes in the shBCL-xL tumors compared to the vehicle group. (E) GSEA revealed alterations in the unfolded protein response, autophagy, and apoptosis pathways in sh–BCL-xL tumors compared to the vector group. All error bars represent means ± SD. **P < 0.01 and ****P < 0.0001.

Article Snippet: To colocate proteins within individual cells, we used conjugated antibodies for LC3B (ab225383 and ab225382), LAMP1 (ab302684), and NF- κB (ab190589 and ab214846) from Abcam, USA and a PLAC8 antibody (CSB- CSB- PA873705LC01HU) from CUSABIO, USA.

Techniques: Inhibition, Injection, Stable Transfection, Plasmid Preparation, Quantitative Proteomics

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A and B) In normal colon, PLAC8 immunofluorescence (red) localizes to the apical domain of the differentiated colonic epithelium at the top of crypts. The boxed region in A is magnified in B. Epithelial cells are outlined by CDH1 immunofluorescence (green). (D) In a typical moderately differentiated adenocarcinoma (H&E-stained sections at lower magnification are shown in C), PLAC8 also localizes to the apical domain, but immunoreactivity extends deeper into the neoplastic crypts. (F and H) PLAC8 immunofluorescence is largely detected in the cytoplasm of medullary (F) and mucinous (H) adenocarcinoma. (C, E, and G) Serial H&E-stained sections at lower magnification correspond to similar areas in D, F, and H. In all immunofluorescent panels, DAPI (blue) marks nuclei. Scale bars: 100 μm.

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Immunofluorescence, Staining

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A) Immunoblotting of CRC cell lines shows PLAC8 levels are higher in cells derived from metastatic tumors (SW620 and KM12SM) compared with cells derived from primary tumors (SW480 and KM12C), as well as in more invasive SC cells compared with CC or parental HCA-7 cells. (B) Left: DIC micrographs of 2 distinct types of HCA-7 colonies in 3D collagen culture: cystic clones (CC) that form smooth-edged spheres and spiky clones (SC) that grow as a solid mass with ill-defined borders and multiple protrusions (left panel) and phalloidin staining (right panel). Right: H&E staining of subcutaneous xenograft tumors of CC and SC cells (left panels); PLAC8 immunofluorescence of CC and SC subcutaneous xenograft tumors (right panels). (C) In 3D collagen culture, by immunoblotting, PLAC8 was undetectable in CC cells, but highly expressed in SC cells, and could be efficiently knocked down by 2 shRNAs in SC cells (left panel). PLAC8 knockdown in SC cells significantly decreased colony number in 3D collagen culture, and reduced tumor volume in xenografts (right panel; **P < 0.01). (D) Knockdown of endogenous PLAC8 in the KM12SM CRC cell line significantly reduced tumor volume of xenografts (left graph, *P < 0.05, n = 7). Right panels are representative H&E-stained tissue sections of xenografts. Data in all graphs are presented as mean ± SEM. Scale bars: 100 μm.

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Western Blot, Derivative Assay, Clone Assay, Staining, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A) Whole-mount ISH using an antisense probe against full-length plac8.1 in zebrafish embryos at indicated hours post-fertilization (hpf). Asterisks denote position of animal poles. The arrow and arrowhead denote future dorsal and ventral side, respectively. Scale bar: 200 μm. (B) Confocal immunofluorescent images of whole-mount zebrafish embryos stained with anti-Plac8.1 antibody (green) and Texas red–conjugated phalloidin (red, F-actin). Cartoons on the left of each panel illustrate corresponding stages. The dashed lines correspond to their right section planes. Scale bar: 10 μm. (C) Whole-mount ISH using plac8.1 antisense probe in zebrafish embryos at 4 days post-fertilization (dpf). Dashed line indicates approximate position for transverse section shown in inset with strong signal in gut. Scale bars: 250 μm. (D) Cryosections through the gut of embryos at 4 dpf were stained with anti-Plac8.1 antibody (green), Texas red–conjugated phalloidin (red, F-actin), and TO-PRO-3 (blue, DNA). Scale bar: 10 μm.

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A) Left: Representative micrographs of control or plac8.1 RNA–injected zebrafish embryo. Horizontal white lines mark the deep cell margin. Middle: ntl ISH. The vertical white lines denote midline tissues, and the red arrowhead indicates the cluster of dorsal forerunner (df) cells separated from the deep cell margin. Right: Micrographs of control and plac8.1 RNA–injected embryos at 3 days post-fertilization (dpf), with ventral view of eyes shown in insets. Scale bars: 200 μm (7.5 hpf); 500 μm (3 dpf); 100 μm (insets). (B) Representative micrographs of a cell cluster with 3 types of membrane junctions: those shared by 2 wild-type cells (white arrows); those shared by 2 Plac8.1-EGFP–overexpressing cells (cyan arrow); and hybrid membrane junctions (green arrows). Scale bars: 10 μm. (C) Quantification of membrane Cdh1 intensity at different membrane junctions. **P < 0.01, ANOVA and subsequent pair-wise t test. (D and E) Quantification of total speed and net speed of lateral mesodermal cells. **P < 0.01, t test. (F) Left: Representative paths of lateral mesodermal cells traveling during time lapse in control and Plac8.1-EGFP–overexpressing embryos. Right: Orientations of the long axes of lateral mesodermal cells are plotted with length/width ratio (LWR) expressed as mean ± SEM. P > 0.05, Mann-Whitney U test. (G) qRT-PCR of cdh1 expression in Plac8.1-overexpressing and control embryos. Data are presented as mean ± SEM (P > 0.05, t test). (H) Immunoblotting of Cdh1 levels in control and Plac8.1-overexpressing embryos at 50% epiboly. Normalized levels are shown as mean ± SEM (P < 0.01, t test).

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Injection, MANN-WHITNEY, Quantitative RT-PCR, Expressing, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A) 2D invasive capacity was assessed by a magnetically attachable stencil invasion assay. After removal of a magnetically attachable stencil, movement of parental HCA-7 and PLAC8-overexpressing HCA-7 (HCA-7P8) cells was monitored over 24 hours by time-lapse microscopy. HCA-7 cells moved as a common front, whereas HCA-7P8 cell movement was uneven and cells detached. Scale bars: 100 μm. (B) At 8 and 24 hours, static images were taken and quantified by 2 parameters (deviation ratio and number of detached cells per field), based on 3 independent experiments performed in triplicate. Both parameters were significantly greater in PLAC8-overexpressing cells. Data are presented as mean ± SD. *P < 0.05; **P < 0.01. (C) After 15 days in 3D collagen culture, PLAC8 and CDH1 immunofluorescence were largely cytosolic in HCA-7 cells overexpressing PLAC8 (HCA-7P8). The boxed regions in the upper panels are magnified in the lower panels. Scale bars: 50 μm.

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Invasion Assay, Time-lapse Microscopy, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A) Total and activated EMT-related kinases were assessed by immunoblotting. Although both p-ERK1 (slower migrating, higher band) and p-ERK2 (faster migrating, lower band) were increased in HCA-7P8 cells, ERK2 phosphorylation was much stronger. Total ERK1/2 proteins were expressed at similar levels in HCA-7 and HCA-7P8 cells. Total AKT and p-AKT were slightly increased, whereas total SRC and p-SRC (Y416) were unchanged in PLAC8-overexpressing HCA-7 cells. (B) Immunoblotting of HCA-7P8 cells infected with nontargeting shRNA vector control (CTL), or shRNAs targeting ERK1 or ERK2, respectively. (C) Representative DIC images from shERK1 and shERK2 cells grown on coverslips for 5 days showed that knockdown of ERK2, but not ERK1, resulted in reversion to smooth-edged colonies similar to parental HCA-7 cells. Scale bar: 500 μm. (D) Representative immunofluorescent images from shERK1 and shERK2 cells showed that knockdown of ERK2, but not ERK1, led to restoration of cell surface CDH1 and markedly reduced CDH3, VIM, and ZEB1. Scale bars: 50 μm.

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Western Blot, Infection, shRNA, Plasmid Preparation

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A) PLAC8 inhibited DUSP6 phosphatase activity in vitro. DUSP6 phosphatase activity was measured by fluorescent intensity of the substrate 3-O-methylfluorescein phosphate. Fluorescent intensity increased over time in control samples (black squares). Addition of purified MBP-tagged mouse PLAC8 protein significantly reduced DUSP6 phosphatase activity (blue circles). However, addition of MBP itself did not significantly affect the activity. Phosphatase inhibitor cocktail was added as a positive control to completely abolish the activity (red squares). Data are presented as mean ± SEM from 4 independent experiments. *P < 0.05, ANOVA followed by t test. (B) Coimmunoprecipitation shows interaction between PLAC8 and DUSP6. HEK293T cells were transfected with the plasmids as labeled. PLAC8 was coimmunoprecipitated with an anti-Myc antibody from cells expressing Myc-tagged DUSP6, but not from cells expressing only PLAC8.

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Activity Assay, In Vitro, Purification, Positive Control, Transfection, Labeling, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A) Control SC cells (CTL) and SC cells stably expressing PLAC8 shRNAs (sh1 and sh2) were subcutaneously injected into athymic nude mice. After 4 weeks, control SC cells formed less well-differentiated tumors with minimal CDH1 immunoreactivity (brown) at the membrane (left). In contrast, SC cells expressing PLAC8 shRNAs formed glandular tumors with large cysts with CDH1 immunoreactivity (brown) observed at the membrane (middle and right). Black box fields are magnified in dashed boxes. Scale bars: 100 μm (top), 20 μm (bottom). (B) Immunoblotting of p-ERK1 (higher band) and p-ERK2 (lower band) in xenograft tumors from control SC cells (CTL, left 3 lanes) and from SC cells stably expressing PLAC8 shRNAs (right 3 lanes). ACTB was used as a loading control.

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Stable Transfection, Expressing, Injection, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

doi: 10.1172/JCI71103

Figure Lengend Snippet: (A) Composite view of PLAC8 (white), CDH1 (red), CDH3 (green), CK (magenta), and VIM (blue) expression. (B) Transition from CDH1- to CDH3-expressing cells from boxed region in A (dotted line). (C) Cells within the CDH3+ (gray) region have cytosolic PLAC8 expression (false-colored, blue). (D) Expression of CK (false-colored, red), VIM (false-colored, green) and cytosolic PLAC8 expression (false-colored, blue) within the CDH3+ (gray) cells. Cells with cytosolic PLAC8 expression and coexpression of CK and VIM are boxed and enlarged in C and D. (E–I) Single-marker expression of panels composited in A. Scale bars in the insets of C and D: 25 μm; other scale bars: 75 μm.

Article Snippet: Expression vectors for human PLAC8 (pcDNA3.1-PLAC8, pcDNA3.1-PLAC8-FLAG, pRetroX-Tight-Pur-PLAC8, and pcDNA3.1-PLAC8-EGFP) were cloned from full-length cDNA (OriGene).

Techniques: Expressing, Marker