Journal: STAR Protocols

Article Title: Protocol to use TopNet for gene regulatory network modeling using gene expression data from perturbation experiments

doi: 10.1016/j.xpro.2022.101737

Figure Lengend Snippet:

Article Snippet: TaqMan probe detecting murine Placenta-specific 8 (Plac8) , Applied Biosystems , Mm00507371_m1.

Techniques: Virus, Retroviral, Plasmid Preparation, Recombinant, Derivative Assay, Reverse Transcription, SYBR Green Assay, Binding Assay, Transgenic Assay, Expressing, Construct, Sequencing, Software

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 1 Expression of plac8 and ERK activation in peripheral blood mononuclear cells of septic patients and normal individuals. A Flow cytometry analysis of peripheral blood CD14+ and CD16+ cell subsets. B Flow cytometry analysis of CD14+ and CD16+ cell subsets. C qRT- PCR analysis of gene expression. D ELISA analysis of cytokine expression. E Western blot analysis of protein expression. F Western blot analysis of protein expression. G Flow cytometry analysis of cell cycle. H Cell proliferation index PI = (S + G2M)/(G0/1 + S + G2M). * indicates P < 0.05 compared to the normal group. Data is presented as mean ± standard deviation. Data analysis was performed using an independent samples t-test, and the experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Expressing, Activation Assay, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 2 Effects of plac8 on peripheral blood mononuclear cell proliferation in septic patients. A qRT-PCR analysis of gene expression. B Western blot analysis of protein band. C Western blot analysis of protein expression. D ELISA analysis of cytokine expression. E Cell proliferation measured by CCK-8 assay, cell proliferation (%) = [OD (treatment group) −OD (blank group)] / [OD (control group) −OD (blank group)] × 100%. * indicates P < 0.05 compared to si-Plac8-NC or Plac8-NC group. Data is presented as mean ± standard deviation. An independent samples t-test was used to compare two groups, and two-way ANOVA was used to compare at different time points. The experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Control, Standard Deviation

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 5 Effects of plac8 on monocyte cell survival and proliferation in the mouse model. A Flow cytometry analysis of CD14+ and CD16+ cell subsets. B Flow cytometry analysis of peripheral blood CD14+ and CD16+ cell subsets. C Western blot analysis of protein band. D Western blot analysis of protein expression. E ELISA analysis of cytokine expression. F Detection of cell proliferation. G Flow cytometry analysis of cell cycle. H Cell proliferation index PI. * indicates P < 0.05 compared to si-Plac8-NC or Plac8-NC group. Data is presented as mean ± standard deviation. Independent samples t-test was used to compare two groups, and two-way ANOVA was used for comparison at different time points. The experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Flow Cytometry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 6 The role and mechanism of Plac8-mediated ERK signaling pathway activation in the proliferation and activation of peripheral blood monocytes in septic patients.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Activation Assay

Journal: Communications Biology

Article Title: PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

doi: 10.1038/s42003-024-07334-8

Figure Lengend Snippet: a Schematic diagram of the RNA sequencing of lung tissues from BLM-treated mice. The lung tissues were collected on day 3 after BLM treatment. (The schematic pictures were downloaded from sciDraw, mouse: 10.5281/zenodo.3925901; syringe: 10.5281/zenodo.4152947). b The volcano plot of mRNA profiling between the lung tissues of BLM-treated and control mice ( n = 6 for the control group, n = 5 for the BLM group). The blue and red dots represent downregulated and upregulated DEGs, respectively. c The heatmap of the expression patterns of DEGs. d Microarray dataset GSE110147 from the Gene Expression Omnibus (GEO) database contained lung tissues of IPF patients and control lung tissues ( n = 11 for the control group, n = 22 for the IPF group). The volcano plot of mRNA profiling between the lung tissues of IPF patients and control donors. The blue and red dots represent downregulated and upregulated differentially expressed genes (DEGs), respectively. e The expression of PLAC8 in the GSE110147 dataset. f Venn diagram exhibited the common gene between the GSE110147 dataset and mRNA-seq from BLM-treated mice. g DEG distribution in mouse chromosomes following BLM treatment. The red and blue bars represent upregulated and downregulated DEGs, respectively. h , i KEGG enrichment analysis and GO enrichment analysis (BP biological processes, CC cellular component, MF molecular function).

Article Snippet: Lung sections were blocked for 15 or 30 min at room temperature with 1% BSA (A602440-0050, sangon) and co-incubated with the primary mouse anti-E-cadherin (60335-1-Ig, Proteintech, Wuhan, China) and rabbit anti-PLAC8 antibody (orb462681, Biorbyt, Cambridge, UK) at 4 °C overnight.

Techniques: RNA Sequencing, Control, Expressing, Microarray, Gene Expression

Journal: Communications Biology

Article Title: PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

doi: 10.1038/s42003-024-07334-8

Figure Lengend Snippet: a , b Hematoxylin and eosin (HE) and Masson’s trichrome staining of lung tissues on day 14 post-BLM administration. c Immunofluorescence analysis of the Surfactant protein C (SPC, green) and TUNEL (red) in lung tissues. The white arrows were pointing to the double positive cells. d The percentages of double TUNEL + SPC+ positive cells in the SPC+ positive cells were determined. e , f Immunoblotting and semi-quantitative analysis of SPC, PLAC8, cleaved caspase-3, LC3, and p62 protein levels in lungs on indicated days after BLM treatment. g Immunofluorescence analysis of the PLAC8 (green) and E-cadherin (red) in lung tissues on day 3 after BLM treatment. h The percentages of double PLAC8 + E-cadherin + positive cells in the E-cadherin+ positive cells were determined. Data represent the mean ± SD (6 mice per group; representative of three independent experiments). Unpaired t -test applied for ( d and h ), and Tukey’s multiple comparison test after the one-way ANOVA was conducted for ( e , f ). * P < 0.05. NS non-significant.

Article Snippet: Lung sections were blocked for 15 or 30 min at room temperature with 1% BSA (A602440-0050, sangon) and co-incubated with the primary mouse anti-E-cadherin (60335-1-Ig, Proteintech, Wuhan, China) and rabbit anti-PLAC8 antibody (orb462681, Biorbyt, Cambridge, UK) at 4 °C overnight.

Techniques: Staining, Immunofluorescence, TUNEL Assay, Western Blot, Comparison

Journal: Communications Biology

Article Title: PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

doi: 10.1038/s42003-024-07334-8

Figure Lengend Snippet: a – d The pmATII cells and A549 cells were treated with 50 μM BLM for 24 h. RT-qPCR analysis and immunoblotting analysis of PLAC8 levels. e , f The mRNA and protein levels of PLAC8 in pmATII cells after infection of adenovirus containing the mouse PLAC8 overexpression vector (oe-PLAC8) or negative control (oe-NC). g , h The mRNA and protein levels of PLAC8 in A549 cells after transfection of PLAC8 overexpression plasmid (OE-PLAC8) or negative control (OE-NC). Immunoblotting analysis of cleaved caspase-3 protein levels in PLAC8-overexpressing pmATII cells ( i ) and A549 cells ( j ) after BLM treatment. Cell apoptosis was detected by TUNEL staining of pmATII cells ( k ) and A549 cells ( l ). Data represent the mean ± SD ( n = 3 per group; representative of three independent experiments. Unpaired t -test applied for ( a and c ), and Tukey’s multiple comparison test after the one-way ANOVA was conducted for ( e – j ). * P < 0.05.

Article Snippet: Lung sections were blocked for 15 or 30 min at room temperature with 1% BSA (A602440-0050, sangon) and co-incubated with the primary mouse anti-E-cadherin (60335-1-Ig, Proteintech, Wuhan, China) and rabbit anti-PLAC8 antibody (orb462681, Biorbyt, Cambridge, UK) at 4 °C overnight.

Techniques: Quantitative RT-PCR, Western Blot, Infection, Over Expression, Plasmid Preparation, Negative Control, Transfection, TUNEL Assay, Staining, Comparison

Journal: Communications Biology

Article Title: PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

doi: 10.1038/s42003-024-07334-8

Figure Lengend Snippet: a , b Transmission electron microscopy (TEM) images of PLAC8-overexpressing pmATII cells and A549 cells following BLM treatment. c A549 cells were transfected with OE-PLAC8 or OE-NC and infected with adenovirus-containing a mCherry- EGFP-LC3B reporter, and cells then were treated with BLM for 24 h. Autophagic flux was measured by counting the LC3 puncta including yellow fluorescence dots (autophagosomes) and red fluorescence dots (autolysosomes) under a microscope. Immunoblotting analysis of LC3 and p62 protein levels in pmATII cells ( d ) and A549 cells ( e ). The PLAC8-overexpressed pmATII cells ( f ) and A549 cells ( h ) were treated with BLM and autophagy inhibitor CQ (20 μM) for 24 h, and cell apoptosis was detected by TUNEL staining. g , i Immunoblotting analysis of cleaved caspase-3 protein levels after BLM and CQ treatment. Data represent the mean ± SD ( n = 3 per group; representative of three independent experiments. Tukey’s multiple comparison test after the one-way ANOVA was conducted for ( c – i ). * P < 0.05. NS non-significant.

Article Snippet: Lung sections were blocked for 15 or 30 min at room temperature with 1% BSA (A602440-0050, sangon) and co-incubated with the primary mouse anti-E-cadherin (60335-1-Ig, Proteintech, Wuhan, China) and rabbit anti-PLAC8 antibody (orb462681, Biorbyt, Cambridge, UK) at 4 °C overnight.

Techniques: Transmission Assay, Electron Microscopy, Transfection, Infection, Fluorescence, Microscopy, Western Blot, TUNEL Assay, Staining, Comparison

Journal: Communications Biology

Article Title: PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

doi: 10.1038/s42003-024-07334-8

Figure Lengend Snippet: a Immunoblotting analysis of p53 protein levels in lungs on days 0, 3, 7, and 14 after BLM treatment. The PLAC8-overexpressed pmATII cells ( b ) and A549 cells ( c ) were treated with BLM for 24 h, and p53 protein levels were detected by immunoblotting analysis. The pmATII cells were infected with oe-PLAC8 with or without oe-TP53 before being treated with BLM. Immunoblotting analysis of cleaved caspase-3 ( d ), LC3, and p62 ( e ) protein levels were detected. f Cell apoptosis was detected by TUNEL staining. A549 cells were transfected with OE-PLAC8 with or without OE-TP53 before being treated with BLM. Immunoblotting analysis of cleaved caspase-3 ( g ), LC3, and p62 ( h ) levels was detected. i Cell apoptosis was detected by TUNEL staining. Data represent the mean ± SD ( n = 3 per group; representative of three independent experiments. Tukey’s multiple comparison test after the one-way ANOVA was conducted for ( a – h ). * P < 0.05. NS non-significant.

Article Snippet: Lung sections were blocked for 15 or 30 min at room temperature with 1% BSA (A602440-0050, sangon) and co-incubated with the primary mouse anti-E-cadherin (60335-1-Ig, Proteintech, Wuhan, China) and rabbit anti-PLAC8 antibody (orb462681, Biorbyt, Cambridge, UK) at 4 °C overnight.

Techniques: Western Blot, Infection, TUNEL Assay, Staining, Transfection, Comparison

Journal: Communications Biology

Article Title: PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

doi: 10.1038/s42003-024-07334-8

Figure Lengend Snippet: a Schematic diagram of the construction of alveolar epithelial type II (ATII)-specific overexpression of PLAC8 under the Surfactant protein B (SPB) promoter and BLM-induced mouse lung fibrosis in mice. (The schematic pictures were downloaded from sciDraw, mouse: doi.org/10.5281/zenodo.3925901; syringe: 10.5281/zenodo.4152947). b , c Hematoxylin and eosin (HE) staining of lung tissues on day 14 post-BLM administration and quantification of lung fibrosis by the Ashcroft score. d , e Masson’s trichrome staining of lung tissues and quantification of lung fibrosis. f Lung/body coefficient was calculated. g Collagen measurement by the hydroxyproline assay in lungs. Immunoblotting analysis of PLAC8 and α-SMA ( h ), collagen I and fibronectin ( i ), p53 ( j ), LC3, and p62 ( k ) in lung tissues of mice. Data represent the mean ± SD (6 mice per group; representative of three independent experiments). Tukey’s multiple comparison test after the one-way ANOVA was conducted for ( c – k ). * P < 0.05.

Article Snippet: Lung sections were blocked for 15 or 30 min at room temperature with 1% BSA (A602440-0050, sangon) and co-incubated with the primary mouse anti-E-cadherin (60335-1-Ig, Proteintech, Wuhan, China) and rabbit anti-PLAC8 antibody (orb462681, Biorbyt, Cambridge, UK) at 4 °C overnight.

Techniques: Over Expression, Staining, Hydroxyproline Assay, Western Blot, Comparison

Journal: Communications Biology

Article Title: PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

doi: 10.1038/s42003-024-07334-8

Figure Lengend Snippet: a Schematic diagram of the COIP/MS analysis of PLAC8-interacting proteins in A549 cells. b Percentages of specific PLAC8-interacting proteins in A549 cells. c KEGG enrichment analysis of PLAC8-interacting proteins. d The proteins enriched in Lysosome, Apoptosis, and Autophagy pathways in KGEE from ( c ). e GO enrichment analysis of PLAC8-interacting proteins. f The proteins enriched in Autophagy, autophagosome maturation, lysosome organization, and proteasome-mediated ubiquitin-dependent protein catabolic process pathways in GO analysis of BP from ( e ). g The proteins enriched in p53 binding, DNA-binding transcription factor binding, Ubiquitination-like modification-dependent protein binding, and Ubiquitination-dependent-protein binding in GO analysis of MF from ( e ). h Protein-protein interaction (PPI) network analysis of protein from ( g ) from the STRING database ( https://cn.string-db.org/ ).

Article Snippet: Lung sections were blocked for 15 or 30 min at room temperature with 1% BSA (A602440-0050, sangon) and co-incubated with the primary mouse anti-E-cadherin (60335-1-Ig, Proteintech, Wuhan, China) and rabbit anti-PLAC8 antibody (orb462681, Biorbyt, Cambridge, UK) at 4 °C overnight.

Techniques: Ubiquitin Proteomics, Binding Assay, Modification, Protein Binding

Journal: Communications Biology

Article Title: PLAC8 attenuates pulmonary fibrosis and inhibits apoptosis of alveolar epithelial cells via facilitating autophagy

doi: 10.1038/s42003-024-07334-8

Figure Lengend Snippet: a Coimmunoprecipitation (COIP) verified the interaction among PLAC8, p53, VCP, UFD1, and NPLOC4 in BLM-treated cells. b Protein-protein interaction (PPI) network analysis of PLAC8, p53, VCP, UFD1, and NPLOC4 in ATII cells based on STRING database, COIP/MS analysis, and COIP experimentally determined. c A549 cells were transduced with OE-PLAC8 and incubated with MG132 and BLM, the ubiquitinated p53 was detected. d The p53 degradation was measured in PLAC8 overexpressing cells after incubation with CHX and BLM. e Cells were transfected with OE-PLAC8 and siRNA against VCP (siVCP) or UFD1 (siUFD1) and then treated with MG132 and BLM. The accumulation of ubiquitinated p53 was detected. f , g Immunoblotting analysis of the p53 expression and degradation in OE-PLAC8 and siVCP transfected cells. h Immunoblotting analysis of LC3 and p62 in A549 cells. i Cell apoptosis was detected by TUNEL staining. Data represent the mean ± SD ( n = 3 per group; representative of three independent experiments). Tukey’s multiple comparison test after the one-way ANOVA was conducted for ( f and h ), Sidak’s test multiple comparison test two-way ANOVA was conducted for ( d and g ). * P < 0.05. NS non-significant.

Article Snippet: Lung sections were blocked for 15 or 30 min at room temperature with 1% BSA (A602440-0050, sangon) and co-incubated with the primary mouse anti-E-cadherin (60335-1-Ig, Proteintech, Wuhan, China) and rabbit anti-PLAC8 antibody (orb462681, Biorbyt, Cambridge, UK) at 4 °C overnight.

Techniques: Transduction, Incubation, Transfection, Western Blot, Expressing, TUNEL Assay, Staining, Comparison

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

Journal: Journal of Cellular and Molecular Medicine

Article Title: PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

doi: 10.1111/jcmm.14578

Figure Lengend Snippet: PLAC8 is relatively up‐regulated in breast cancer. A, The mRNA expression of PLAC8 is increased in breast cancer tissues compared with adjacent tissues, as determined by qPCR. The mRNA expression was normalized to adjacent tissues. The error bars correspond to the means ± SD. (B) IHC staining showed the expression of PLAC8 in breast cancer and adjacent tissues of patients. 20× and 40×; the scale bar represents 100 and 50 μm, respectively. C, The PLAC8 protein expression levels of breast cancer cell lines (HCC1937, T47D, MCF‐7, Hs‐578T, SK‐BR‐3, BT549, BCAP‐37 and MDA‐MB‐231). D, The subcellular localization of PLAC8 in BCAP‐37 and T47D cells was detected by immunofluorescence analysis. An enlarged view of the boxed area from each group confirms the presence of PLAC8‐positive cells. The scale bar represents 10 μm. The values are presented as the means ± SD ** P < 0.01

Article Snippet: Short interfering RNAs targeting PLAC8 (Si‐PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

doi: 10.1111/jcmm.14578

Figure Lengend Snippet: PLAC8 promotes cell proliferation in vitro . A and B, Bcap‐37 and T47D cells were transfected with PLAC8 siRNA and/or the PLAC8 overexpression plasmid, and the interference effect of siRNA or plasmid was determined by Western blotting (upper) 72 h after transfection and qRT‐PCR (bottom) 48 h after transfection. The expression was quantified and normalized to GAPDH. The error bars correspond to the means ± SD. (C) Cell proliferation assays were performed to determine the cell viability for Bcap‐37 and T47D cells transfected with PLAC8 siRNA or plasmid. The error bars correspond to the means ± SD. The values are presented as the means ± SD * P < 0.05; ** P < 0.01; NS, not significant

Article Snippet: Short interfering RNAs targeting PLAC8 (Si‐PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China).

Techniques: In Vitro, Transfection, Over Expression, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

doi: 10.1111/jcmm.14578

Figure Lengend Snippet: PLAC8 knockdown significantly inhibits cell migration and invasion and suppresses the EMT phenotype. A, Wound‐healing assays were conducted in Bcap‐37 or T47D cells transfected with PLAC8 siRNA or the PLAC8 overexpression plasmid. Migration distance was measured at 0 and 24 h after cells were scratched, Bcap‐37 Si‐NC or T47D vector cells were used as controls. The error bars correspond to the means ± SD. B, Representative images show that cell migration (upper) and invasion (bottom) increased in the PLAC8‐overexpressing cells. The error bars correspond to the means ± SD. C, Western blot analysis of the relative expression of EMT‐related markers in Bcap‐37 or T47D cells 72 h after transfection. The expression was quantified and normalized to GAPDH. The error bars correspond to the means ± SD. The values are presented as the means ± SD * P < 0.05; ** P < 0.01; NS, not significant

Article Snippet: Short interfering RNAs targeting PLAC8 (Si‐PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Migration, Transfection, Over Expression, Plasmid Preparation, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

doi: 10.1111/jcmm.14578

Figure Lengend Snippet: PLAC8 inhibits apoptosis both in vivo and vitro. A and B, Bcap‐37 and T47D cells were stained with Annexin V/PI or Tunnel solution, and the percentage of apoptotic cells was determined using a flow cytometer 48 h after transfection. An enlarged view of the boxed area from each group confirms the presence of TUNEL‐positive cells. The values are presented as the means ± SD ** P < 0.01; NS, not significant

Article Snippet: Short interfering RNAs targeting PLAC8 (Si‐PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China).

Techniques: In Vivo, Staining, Flow Cytometry, Transfection, TUNEL Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

doi: 10.1111/jcmm.14578

Figure Lengend Snippet: PLAC8 inhibits cell proliferation by influencing apoptosis in vivo. A, Western blot analysis of the relative expression of apoptosis‐related markers 72 h after transfection. The expression was quantified and normalized to GAPDH. The error bars correspond to the means ± SD. B, Analysis of the cell cycle in Bcap‐37 or T47D cells 48 h after PLAC8 siRNA or plasmid transfection, determined using a flow cytometer. The error bars correspond to the means ± SD. The values are presented as the means ± SD * P < 0.05; ** P < 0.01; NS, not significant

Article Snippet: Short interfering RNAs targeting PLAC8 (Si‐PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China).

Techniques: In Vivo, Western Blot, Expressing, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

doi: 10.1111/jcmm.14578

Figure Lengend Snippet: PLAC8 enhances tumour growth in vitro and in vivo by decreasing apoptosis. A, Tumour volume (measured twice a week) and tumour weight (day 18) were compared between the PLAC8‐overexpressing and vector groups, which showed tumour volumes and weights that were comparable to those observed in the vector group. The tumour volume was calculated using the following equation: V = (width 2 × length)/2. The error bars correspond to the means ± SD. B, IHC staining showed the expression of PLAC8, Ki67 and cleaved caspase‐3. 20× and 40×; the scale bar represents 100 and 50 μm, respectively. C, Assessment of apoptosis by TUNEL assays of tumours from mice in the PLAC8 overexpression and vector groups on day 18. The scale bar represents 20 μm. An enlarged view of the boxed area from each group confirms the presence of TUNEL‐positive cells. The values are presented as the means ± SD * P < 0.05; ** P < 0.01; NS, not significant

Article Snippet: Short interfering RNAs targeting PLAC8 (Si‐PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China).

Techniques: In Vitro, In Vivo, Plasmid Preparation, Immunohistochemistry, Expressing, TUNEL Assay, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

doi: 10.1111/jcmm.14578

Figure Lengend Snippet: PLAC8 knockdown induces apoptosis and affects cell proliferation by inhibiting the PI3K/AKT/NF‐κB pathway. A, Western blot analysis of Bcap‐37 or T47D cells 72 h after PLAC8 siRNA or plasmid transfection. Scramble RNA or vector plasmid was used as negative controls. The levels of PI3K/AKT/NF‐κB pathway‐related markers were measured. Protein expression was quantified and normalized to GAPDH. The error bars correspond to the means ± SD. (B) Cells were transfected for 48 h and subsequently cultured with or without LY294002 (10 μm) for 24 h. Western blot analysis showing changes in the levels of PI3K pathway‐related markers. Protein expression was quantified and normalized to GAPDH. The error bars correspond to the means ± SD. The values are presented as the means ± SD * P < 0.05; ** P < 0.01; NS, not significant

Article Snippet: Short interfering RNAs targeting PLAC8 (Si‐PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Western Blot, Plasmid Preparation, Transfection, Expressing, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: PLCA8 suppresses breast cancer apoptosis by activating the PI3k/AKT/NF‐κB pathway

doi: 10.1111/jcmm.14578

Figure Lengend Snippet: The relationship between PLAC8 expression and clinicopathological characteristics in breast cancer

Article Snippet: Short interfering RNAs targeting PLAC8 (Si‐PLAC8) and a scrambled control siRNA were designed and purchased from RiboBio (Guangzhou, China).

Techniques: Expressing