Journal: Journal of Translational Medicine

Article Title: AZGP1 deficiency promotes angiogenesis in prostate cancer

doi: 10.1186/s12967-024-05183-x

Figure Lengend Snippet: AZGP1 has no impact on cell proliferation in PCa cells. A Transcript expression levels of AZGP1 are higher in LNCaP, C42B, and 22Rv1 cell lines compared to PC3 and DU145 cells, as determined by RT-qPCR. B Western blot analysis of protein levels of AZGP1 are higher in LNCaP, C42B, and 22Rv1 cell lines compared to PC3 and DU145 cells. C Knockout of AZGP1 in LNCaP sg-AZGP1-T1 and sg-AZGP1-T2 cells showed no significant effect on cell proliferation. D Western blot confirming the knockout of AZGP1 in LNCaP sg-AZGP1-T1 and sg-AZGP1-T2 cells. Blot membrane was incubated with secondary antibody overnight. E Overexpression of AZGP1 in LNCaP AZGP1-OV cells did not impact cell proliferation. F Western blot confirming increased AZGP1 protein expression in LNCaP AZGP1-OV cells. The blot membrane was incubated with secondary antibody for 1 h. G Knockout of AZGP1 had no effect on colony formation in LNCaP cells. H Quantification of colony formation in control and AZGP1 knockout sg-AZGP1-T1 and sg-AZGP1-T2 LNCaP cells. I Overexpression of AZGP1 had no effect on colony formation in LNCaP cells. J Quantification of colony formation in LNCaP control and AZGP1-OV cells. K Knockout of AZGP1 had no significant effect on cell proliferation in LuCaP147 cells. L Western blot confirming the knockout of AZGP1 in LuCaP147 sg-AZGP1-T1 and sg-AZGP1-T2 cells. Blot membrane was incubated with secondary antibody overnight. M Overexpression of AZGP1 did not affect cell proliferation in LuCaP147 cells. N Western blot confirming the overexpression of AZGP1 in LuCaP147 AZGP1-OV cells. Blot membrane was incubated with secondary antibody for 1 h. Note: the exposure time for AZPG1 knockout cells (LNCaP control, sg-AZGP1-T1 and sg-AZGP1-T2 cells, LuCaP147 control, sg-AZGP1-T1 and sg-AZGP1-T2 cells,) and AZGP1 overexpression cells (LNCaP control and AZGP1-OV, LuCaP147 control and AZGP1-OV cells) are different

Article Snippet: For antigen retrieval, the tissue sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20 min, and slides were blocked in 5% goat serum and 5% BSA in PBS for 1 h. After that, the sections were incubated with a primary antibody AZGP1 (TA8121185, 1:100 dilution, OriGene Technologies, Inc., MD, USA), CD31(1:100, ab182981, Abcam, Boston, MA, USA) in PBS containing 0.5% BSA overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Knock-Out, Membrane, Incubation, Over Expression, Control

Journal: Journal of Translational Medicine

Article Title: AZGP1 deficiency promotes angiogenesis in prostate cancer

doi: 10.1186/s12967-024-05183-x

Figure Lengend Snippet: AZGP1 does not affect cell migration or invasion in PCa cells. A Wound-healing assay of PC3 vector control and PC3 AZGP1-OV cells shows no effect on cell migration. C Quantification of migration in PC3 vector control and PC3 AZGP1-OV cells. B Western blot confirming the overexpression of AZGP1 in PC3 AZGP1-OV cells. D Wound-healing assay of DU145 vector control and DU145 AZGP1-OV demonstrating no effect of AZGOP1 on DU145 cell migration. F Quantification of migration ability in DU145 vector control and DU145 AZGP1-OV cells. E Western blot confirming the overexpression of AZGP1 in DU145 AZGP1-OV cells. G Transwell migration assay PC3 vector control and PC3 AZGP1-OV demonstrating does not affect PC3 cell migration. H Quantification of migration ability in PC3 vector control and PC3 AZGP1-OV cells. I Transwell invasion assay of PC3 vector control and DU145 AZGP1-OV showing that overexpression of AZGP1 does not affect PC3 cell invasion. J Quantification of invasion ability in PC3 control and AZGP1-OV cells

Article Snippet: For antigen retrieval, the tissue sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20 min, and slides were blocked in 5% goat serum and 5% BSA in PBS for 1 h. After that, the sections were incubated with a primary antibody AZGP1 (TA8121185, 1:100 dilution, OriGene Technologies, Inc., MD, USA), CD31(1:100, ab182981, Abcam, Boston, MA, USA) in PBS containing 0.5% BSA overnight at 4 °C.

Techniques: Migration, Wound Healing Assay, Plasmid Preparation, Control, Western Blot, Over Expression, Transwell Migration Assay, Transwell Invasion Assay

Journal: Journal of Translational Medicine

Article Title: AZGP1 deficiency promotes angiogenesis in prostate cancer

doi: 10.1186/s12967-024-05183-x

Figure Lengend Snippet: Loss of AZGP1 promotes stromal cell hyperplasia in the prostate of AZGP1 −/− mice. A Schematic representation of endogenous AZGP1, AZGP1 knockout, and targeting construct. B Genotype analysis of AZGP1 of wild-type, heterozygous AZGP1 ± and homozygous AZGP1 −/− mice. C Immunofluorescence staining of CD31 and its corresponding quantification of microvessle counts per area in prostate excluding SV from wild-type and homozygous AZGP1 −/− mice. Increased microvessel density was observed in homozygous AZGP1 −/− mouse prostates. Microvessels were counted from 10 randomly selected 20X microscope fields in the CD31-stained sections. D Hematoxylin and eosin (H&E) staining of the LP lateral prostate, DP dorsal prostate and basal part of SV seminal vesicles from wild-type and homozygous AZGP1 −/− mice suggest that there are no morphological changes in LP and DP. Increased fibrosis were observed at the base of the seminal vesicles as illustrated with sirius red staining

Article Snippet: For antigen retrieval, the tissue sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20 min, and slides were blocked in 5% goat serum and 5% BSA in PBS for 1 h. After that, the sections were incubated with a primary antibody AZGP1 (TA8121185, 1:100 dilution, OriGene Technologies, Inc., MD, USA), CD31(1:100, ab182981, Abcam, Boston, MA, USA) in PBS containing 0.5% BSA overnight at 4 °C.

Techniques: Knock-Out, Construct, Immunofluorescence, Staining, Microscopy

Journal: Journal of Translational Medicine

Article Title: AZGP1 deficiency promotes angiogenesis in prostate cancer

doi: 10.1186/s12967-024-05183-x

Figure Lengend Snippet: Overexpression of AZGP1 inhibits angiogenesis in PC3 and DU145 tumors. A Representative images of PC3 vector control and PC3-AZGP1-OV tumor xenografts. B Calculated tumor volume, and C Tumor weight of PC3 vector control and PC3-AZGP1-OV tumor xenografts. D Quantification of the degree of redness of PC3 vector control and PC3-AZGP1-OV tumor xenografts using python, showing reduced redness in AZGP1-OV PC3 tumors. E CD31 staining showing lower microvessel density in AZGP1-OV PC3 tumors compared to controls. Microvessels were counted from 10 randomly selected high powered microscope fields uon the CD31-stained sections. F Microscopic images showing similar tumor morphology by H & E, validation of AZGP1 overexpression, and decreased number of CD31 staining microvessels in PC3-AZGP1-OV tumors compared to controls. G Representative images of DU145 vector control and DU145-AZGP1-OV tumor xenografts. H Calculated tumor volume, and I tumor weight of control and AZGP1-OV DU145 tumors. J Quantification of tumor redness using Image J, indicating reduced redness in AZGP1-OV DU145 tumors compared to vector control. K DU145 AZGP1-OV tumors displayed lower microvessel density compared to controls. L Microscopic images showing similar tumor morphology by H and E , validation of AZGP1 overexpression, and decreased number of CD31 staining microvessels in DU145-AZGP1-OV tumors compared to controls

Article Snippet: For antigen retrieval, the tissue sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20 min, and slides were blocked in 5% goat serum and 5% BSA in PBS for 1 h. After that, the sections were incubated with a primary antibody AZGP1 (TA8121185, 1:100 dilution, OriGene Technologies, Inc., MD, USA), CD31(1:100, ab182981, Abcam, Boston, MA, USA) in PBS containing 0.5% BSA overnight at 4 °C.

Techniques: Over Expression, Plasmid Preparation, Control, Staining, Microscopy, Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: AZGP1 deficiency promotes angiogenesis in prostate cancer

doi: 10.1186/s12967-024-05183-x

Figure Lengend Snippet: AZGP1 protein expression in PC3 cell lines modulates the angiogenesis pathway. A Volcano plot of proteome of PC3 vector control and PC3-AZGP1-OV shows 1282 significantly modulated proteins (grey dots) and 94 were identified as angiogenesis-related genes (red dots). Proteins illustrated in red demonstrate 29 upregulated genes and 25 downregulated genes that are associated with angiogenesis. B Hypergeometric distribution of angiogenesis genes shows that the identified 54 genes are significantly upregulated or downregulated between PC3 control and AZPG1-OV tumors ( p = 0.014) C Heatmap of the 54 angiogenesis genes in AZGP1-OV PC3 and controls shows clear segregation of the over-expressing and low expressing cells. D Relative protein expression levels of MMP9, E PDCD6, F EphA2 in AZGP1-OV PC3 and control tumors. G Western blot confirming decreased levels of MMP9 and increased levels of PDCD6 and EphA2 proteins, and validation of AZGP1 overexpression in AZGP1-OV PC3 tumors

Article Snippet: For antigen retrieval, the tissue sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20 min, and slides were blocked in 5% goat serum and 5% BSA in PBS for 1 h. After that, the sections were incubated with a primary antibody AZGP1 (TA8121185, 1:100 dilution, OriGene Technologies, Inc., MD, USA), CD31(1:100, ab182981, Abcam, Boston, MA, USA) in PBS containing 0.5% BSA overnight at 4 °C.

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Biomarker Discovery, Over Expression

Journal: Journal of Translational Medicine

Article Title: AZGP1 deficiency promotes angiogenesis in prostate cancer

doi: 10.1186/s12967-024-05183-x

Figure Lengend Snippet: AZGP1 inhibits HUVEC proliferation, migration and tube formation. A AZGP1 slightly inhibits the HUVEC growth after 7-days treatment. B AZGP1 treatment inhibits tube formation ability and node formation of HUVECs. C Quantification of HUVEC nodes counted from 10 hpf. D Diagram summarizing the transwell migration assay. E There were no differences in cells migrating to the bottom of the transwells in AZGP1-treated and untreated HUVECs. However Significantly fewer cells were found in the bottom wells in AZGP1-treated HUVECs. The corresponding quantification of cells being migrated to ( F ) bottom of the transwell and, G the bottom of the wells

Article Snippet: For antigen retrieval, the tissue sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20 min, and slides were blocked in 5% goat serum and 5% BSA in PBS for 1 h. After that, the sections were incubated with a primary antibody AZGP1 (TA8121185, 1:100 dilution, OriGene Technologies, Inc., MD, USA), CD31(1:100, ab182981, Abcam, Boston, MA, USA) in PBS containing 0.5% BSA overnight at 4 °C.

Techniques: Migration, Transwell Migration Assay

Journal: Journal of Translational Medicine

Article Title: AZGP1 deficiency promotes angiogenesis in prostate cancer

doi: 10.1186/s12967-024-05183-x

Figure Lengend Snippet: AZGP1 is involved in angiogenesis in human prostate cancer. A Low expression level of AZGP1 is correlated with poor survival in the PRAD-TCGA database. B Lower expression of AZGP1 is associated with higher Gleason scores in the PRAD-TCGA database. C Decreased expression level of AZGP1 shows strong correlation with poor survival in the MSKCC database. D Lower expression of AZGP1 is associated with higher Gleason scores in the MSKCC database. E GO biological process analysis of the 74 overlapping genes shows significant roles in angiogenesis. F GO molecular function analysis of the 74 overlapping genes. TF: transcription factor G WikiPathways analysis of the 74 genes implicates the VEGF signaling pathway as the possible target of AZGP1 expression modulation in prostate cancer cells. H Representative images of immunofluorescence staining of DAPI, AZGP1, and CD31 in a human tissue microarray (TMA) of localized prostate cancer. I Representative image of a virtual hematoxylin and eosin stain of TMA cores to demonstrate the cancer morphology with superimposed microvessels determined by AI. J Human prostate tumor samples were divided at the median into high and low blood vessel density. AZGP1 expression was significantly higher in the low blood vessel density group compared to the high blood vessel density group. K The expression of AZGP1 is negatively correlated with blood vessel density

Article Snippet: For antigen retrieval, the tissue sections were boiled in 0.01 M citrate buffer (pH 6.0) for 20 min, and slides were blocked in 5% goat serum and 5% BSA in PBS for 1 h. After that, the sections were incubated with a primary antibody AZGP1 (TA8121185, 1:100 dilution, OriGene Technologies, Inc., MD, USA), CD31(1:100, ab182981, Abcam, Boston, MA, USA) in PBS containing 0.5% BSA overnight at 4 °C.

Techniques: Expressing, Immunofluorescence, Staining, Microarray, H&E Stain

Journal: Chest

Article Title: Cigarette Smoking Induces Overexpression of a Fat-Depleting Gene AZGP1 in the Human

doi: 10.1378/chest.08-1024

Figure Lengend Snippet: Immunofluorescence assessment of AZGP1 colocalization with mucin 5AC and chromogranin A. Cytospin preparations of large airway epithelium from a healthy nonsmoker were stained with antibodies against AZGP1, mucin 5AC, and chromogranin A, followed by a Cy5-conjugated secondary antibody for AZGP1 (shown in green) and a Cy3-conjugated antibody for mucin 5AC and chromogranin A (shown in red). Nuclei were stained with DAPI (shown in blue); colocalization of AZGP1 with either mucin 5AC or chromogranin A is shown in yellow. Colocalization of AZGP1 and mucin 5AC (panels A to I). Phase contrast image of a cytospin preparation from a healthy nonsmoker showing large airway epithelial cells (top left, A). Top center, B: cytospin preparation from a healthy nonsmoker (same field as in top left, A), but in the Cy5 channel showing AZGP1 staining of large airway epithelial cells. Top left, C: cytospin preparation from a healthy nonsmoker (same field as in top left, A), but in the Cy3 channel showing mucin 5AC staining. Top middle left, D: cytospin preparation from a healthy nonsmoker (same field as in top left, A) showing colocalization of AZGP1 and mucin 5AC (shown in yellow); nuclei are shown in blue. Top middle center, E: cytospin preparation from a healthy nonsmoker (same field as in top left, A) viewed in phase contrast showing colocalization of AZGP1 and mucin 5AC as well as DAPI nuclear staining. Top middle right, F: cytospin preparation from a healthy nonsmoker (same field as in top left, A) demonstrating very minimal to no autofluorescence of large airway epithelial cells. Middle left, G: cytospin preparation from a healthy nonsmoker viewed in phase contrast showing a secretory airway epithelial cell. Middle center, H: cytospin preparation from a healthy nonsmoker (same field as in middle left, G) viewed with the Cy3 and Cy5 channels (stained with both AZGP1 and mucin 5AC antibodies), showing a secretory airway epithelial cell positive for mucin 5AC but negative for AZGP1; nuclei are shown in blue. Middle right, I: cytospin preparation from a healthy nonsmoker (same field as in middle left, G) showing an example of AZGP1-negative mucin 5AC-positive secretory airway epithelial cell shown as three-channel fluorescent view (including phase view). Colocalization of AZGP1 and chromogranin A (panels J–O). Bottom middle left, J: phase contrast view of cytospin preparation from a healthy nonsmoker showing airway epithelial cell. Bottom middle center, K: cytospin preparation from a healthy nonsmoker (same field as in bottom middle left, J) showing an example of colocalization AZGP1 and chromogranin A in an airway epithelial cell; nucleus is shown in blue. Bottom middle right, L: cytospin preparation from a healthy nonsmoker (same field as in bottom middle left, J) showing an example of colocalization of AZGP1 and chromogranin A in an airway epithelial cell shown as three-channel fluorescent view (including phase view). Bottom left, M: phase contrast view of cytospin preparation from a healthy nonsmoker showing airway epithelial cell. Bottom center, N: cytospin preparation from a healthy nonsmoker (same field as in bottom left, M) showing an example of colocalization AZGP1 and chromogranin A in an airway epithelial cell; nucleus is shown in blue. Bottom right, O: cytospin preparation from a healthy nonsmoker (same field as in bottom left, M) showing an example of colocalization of AZGP1 and chromogranin A in an airway epithelial cell shown as three-channel fluorescent view (including phase view). Bar = 0.01 mm. Original magnification for all panels is ×60.

Article Snippet: Following incubation with the primary antibodies, goat antirabbit Cy5 (Jackson ImmunoResearch) was used as a secondary antibody for AZGP1, and goat antimouse Cy3 (Jackson ImmunoResearch) was used as a secondary antibody for chromogranin A and mucin 5AC.

Techniques: Immunofluorescence, Staining

Journal: Chest

Article Title: Cigarette Smoking Induces Overexpression of a Fat-Depleting Gene AZGP1 in the Human

doi: 10.1378/chest.08-1024

Figure Lengend Snippet: Quantification of AZGP1-positive cells in healthy nonsmokers and healthy smokers. Cytospin preparations of large airway epithelial cells from five nonsmokers and six smokers were stained with rabbit antihuman AZGP1 or rabbit IgG as control. Goat antirabbit Cy3 was used as a secondary antibody. Nuclei were counterstained with DAPI. The percentage of AZGP1-positive cells was calculated as the number of AZGP1-positive cells per field/total number of nuclei per field in 10 random ×60 fields for each subject. Representative low-power (original ×20) staining of specimens from one healthy nonsmoker and one healthy smoker are shown. Top left, A: healthy nonsmoker, rabbit IgG1 isotype control. Top center, B: healthy smoker, rabbit IgG1 isotype control. Bottom left, C: healthy nonsmoker, rabbit antihuman AZGP1 antibody. Bottom center, D: healthy smoker, rabbit antihuman AZGP1 antibody. Bar = 0.01 mm. Right, E: quantification of AZGP1-positive cells; the ordinate shows percentage of AZGP1-positive cells; the abscissa shows the different groups (n = 5 healthy nonsmokers, n = 6 healthy smokers). Error bars represent the SD.

Article Snippet: Following incubation with the primary antibodies, goat antirabbit Cy5 (Jackson ImmunoResearch) was used as a secondary antibody for AZGP1, and goat antimouse Cy3 (Jackson ImmunoResearch) was used as a secondary antibody for chromogranin A and mucin 5AC.

Techniques: Staining, Control

Journal: Clinical Proteomics

Article Title: Proteins from formalin-fixed paraffin-embedded prostate cancer sections that predict the risk of metastatic disease

doi: 10.1186/s12014-015-9096-3

Figure Lengend Snippet: Representative Western blot images of matched tumor and control regions from patients with low or high risk of early biochemical failure. Proteins were separated by 1D-PAGE, transferred to Hybond-LFP membrane and probed for ANXA2, AZGP1, PSA and PRDX1, plus actin as a loading control. Detection with AlexaFluor-labelled second antibodies was captured using a FLA-5100 scanner

Article Snippet: Protein extracts (15–20 µg protein) were resolved by 4–12 % 1D-PAGE and transferred to Hybond-LFP membranes (GE Healthcare) for 1 h at 30 V. The membranes were blocked in PBS containing 0.1 % Tween-20 and 0.25 % gelatin for 90 min at room temperature and then probed overnight at room temperature with the following antibodies: ANXA2 (C-10, mouse monoclonal, Santa Cruz, sc-28385) 1:100; PSA (C-19, goat polyclonal, Santa Cruz, sc-7638) 1:200; PRDX1 (N-19, goat polyclonal, Santa Cruz, sc-7381) 1:200; AZGP1 (H123, rabbit polyclonal, Santa Cruz, sc-11358) 1:200.

Techniques: Western Blot