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Cusabio plac8 antibody

Fig. 1. Induction of <t>PLAC8</t> 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.

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1) Product Images from "Interaction between NF-κB and PLAC8 impairs autophagy providing a survival advantage to prostate cells transformed by cadmium."

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

Journal: Science advances

doi: 10.1126/sciadv.adv8640

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

Image Search Results

(A) PLAC8 IHC in tissue microarray (78 tumor/normal pairs). CON: normal adjacent tissue; T-1/T-2/T-3: representative PLAC8 protein highly expression tumor tissues. (B) Comparative IHC scores between tumor and normal tissues. (C) Patients were stratified into subgroups based on PLAC8 IHC scoring criteria and survival status, followed by Kaplan-Meier survival curve analysis with log-rank testing. (D) The colorectal cancer chemoprevention model was established through sequential administration of azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice. (E) PLAC8 protein expression dynamics were analyzed throughout the tumorigenesis process. (Student’s t-test for comparisons).

Journal: Frontiers in Immunology

Article Title: Targeting the CCL28-STAT3-PLAC8 axis to suppress metastasis and remodel tumor microenvironment in colorectal cancer

doi: 10.3389/fimmu.2025.1610540

Figure Lengend Snippet: (A) PLAC8 IHC in tissue microarray (78 tumor/normal pairs). CON: normal adjacent tissue; T-1/T-2/T-3: representative PLAC8 protein highly expression tumor tissues. (B) Comparative IHC scores between tumor and normal tissues. (C) Patients were stratified into subgroups based on PLAC8 IHC scoring criteria and survival status, followed by Kaplan-Meier survival curve analysis with log-rank testing. (D) The colorectal cancer chemoprevention model was established through sequential administration of azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice. (E) PLAC8 protein expression dynamics were analyzed throughout the tumorigenesis process. (Student’s t-test for comparisons).

Article Snippet: The sections were then incubated with a primary anti-PLAC8 antibody (Proteintech, 12284-1-AP, 1:100) overnight at 4 °C, followed by a 30-minute incubation at room temperature with a biotinylated secondary antibody.

Techniques: Microarray, Expressing

(A) PLAC8 expression in four CRC cell lines. (B, C) PLAC8 overexpression and knockdown conducted in lower and higher PLAC8 expression CRC cell lines. (D, E) Impact of PLAC8 expression changes on cell clonogenicity and proliferation. (F) Transwell assay assessing PLAC8 expression on migration of CRC cell lines. (G) TCGA-based bioinformatic analysis to explore PLAC8 involvement in platinum resistance. (H) Impact of PLAC8 expression changes on oxaliplatin resistance (Student’s t-test for comparisons; *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively; GSVA for enrichment).

Journal: Frontiers in Immunology

Article Title: Targeting the CCL28-STAT3-PLAC8 axis to suppress metastasis and remodel tumor microenvironment in colorectal cancer

doi: 10.3389/fimmu.2025.1610540

Figure Lengend Snippet: (A) PLAC8 expression in four CRC cell lines. (B, C) PLAC8 overexpression and knockdown conducted in lower and higher PLAC8 expression CRC cell lines. (D, E) Impact of PLAC8 expression changes on cell clonogenicity and proliferation. (F) Transwell assay assessing PLAC8 expression on migration of CRC cell lines. (G) TCGA-based bioinformatic analysis to explore PLAC8 involvement in platinum resistance. (H) Impact of PLAC8 expression changes on oxaliplatin resistance (Student’s t-test for comparisons; *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively; GSVA for enrichment).

Techniques: Expressing, Over Expression, Knockdown, Transwell Assay, Migration

(A) TCGA-based bioinformatic analysis to explore PLAC8 involvement in chemokine signaling. (B) Effect of PLAC8 expression changes on inflammation and cytokine-related signaling pathways. (C) Reversal by AKT pathway activator SC79, the reduction in migration and invasion abilities induced by PLAC8 knockdown. (D) Reversal by AKT inhibitor capivasertib, the enhanced migration and invasion abilities of colorectal cancer cells induced by PLAC8 overexpression. (E) Effects of PLAC8 modulation and AKT pathway activator and inhibitor on EMT-related protein expression. (GSVA for enrichment).

Journal: Frontiers in Immunology

Article Title: Targeting the CCL28-STAT3-PLAC8 axis to suppress metastasis and remodel tumor microenvironment in colorectal cancer

doi: 10.3389/fimmu.2025.1610540

Figure Lengend Snippet: (A) TCGA-based bioinformatic analysis to explore PLAC8 involvement in chemokine signaling. (B) Effect of PLAC8 expression changes on inflammation and cytokine-related signaling pathways. (C) Reversal by AKT pathway activator SC79, the reduction in migration and invasion abilities induced by PLAC8 knockdown. (D) Reversal by AKT inhibitor capivasertib, the enhanced migration and invasion abilities of colorectal cancer cells induced by PLAC8 overexpression. (E) Effects of PLAC8 modulation and AKT pathway activator and inhibitor on EMT-related protein expression. (GSVA for enrichment).

Techniques: Expressing, Protein-Protein interactions, Migration, Knockdown, Over Expression

(A) Pearson correlation analysis between PLAC8 expression and the top five inflammatory cytokine pathways gene based on the TCGA database. (B) Regulation of PLAC8 mRNA expression by Ruxolitinib (STAT3 inhibitor), CXCL1, and CCL28. (C) Regulation of STAT3 signaling and PLAC8 protein expression by Ruxolitinib and CCL28. (D) Effects of combined PLAC8 knockdown and CCL28 treatment, or STAT3 inhibition and PLAC8 overexpression, on CRC cell migration. (E, F) Validation of STAT3 binding to the PLAC8 promoter by CUT&Tag. (**, and *** represent P < 0.01, and P < 0.001, respectively).

Journal: Frontiers in Immunology

Article Title: Targeting the CCL28-STAT3-PLAC8 axis to suppress metastasis and remodel tumor microenvironment in colorectal cancer

doi: 10.3389/fimmu.2025.1610540

Figure Lengend Snippet: (A) Pearson correlation analysis between PLAC8 expression and the top five inflammatory cytokine pathways gene based on the TCGA database. (B) Regulation of PLAC8 mRNA expression by Ruxolitinib (STAT3 inhibitor), CXCL1, and CCL28. (C) Regulation of STAT3 signaling and PLAC8 protein expression by Ruxolitinib and CCL28. (D) Effects of combined PLAC8 knockdown and CCL28 treatment, or STAT3 inhibition and PLAC8 overexpression, on CRC cell migration. (E, F) Validation of STAT3 binding to the PLAC8 promoter by CUT&Tag. (**, and *** represent P < 0.01, and P < 0.001, respectively).

Techniques: Expressing, Knockdown, Inhibition, Over Expression, Migration, Biomarker Discovery, Binding Assay

Schematic illustrating how the CCL28-STAT3-PLAC8 axis promotes EMT and enhances CRC invasion and metastasis via AKT signaling activation.

Journal: Frontiers in Immunology

Article Title: Targeting the CCL28-STAT3-PLAC8 axis to suppress metastasis and remodel tumor microenvironment in colorectal cancer

doi: 10.3389/fimmu.2025.1610540

Figure Lengend Snippet: Schematic illustrating how the CCL28-STAT3-PLAC8 axis promotes EMT and enhances CRC invasion and metastasis via AKT signaling activation.

Techniques: Activation 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. 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.

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.

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.

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.

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.

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

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