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