rabbit polyclonal anti homer3  (Atlas Antibodies)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

doi: 10.1186/s13046-021-01988-6

Figure Lengend Snippet: Identification of targetable glycoproteins in bladder cancer supported by bioinformatics analysis. A ) Venn Diagram showing glycoproteins identified by targeted glycoproteomics focusing on sialylated T antigens in different cell models. A total of 903 glycoproteins were identified by mass spectrometry; however, only 28% were common to both cell lines, suggesting considerable distinct glycoproteomes. B ) Venn diagram for glycoproteins overexpressed in MIBC according to the Oncomine (RNAseq) database. Glycoproteins were comprehensively matched with transcriptomic data in Oncomine to sort species potentially upregulated in MIBC in relation to non-pathological urothelium. Glycoproteins upregulated in both NMIBC and MIBC were also included for downstream analysis. 11% (95/903 assignments) were upregulated in MIBC, with the cell lines showing again very distinct and unique glycoproteomes. These glycoproteins were elected for specificity evaluation. C ) Gene expression (Oncomine, RNASeq) vs protein levels (Human Protein Atlas; IHQ) for glycoproteins showing gene upregulation in MIBC. This diagram shows that 95 out of the 903 sorted glycoproteins were detected in MIBC, 81% of which were also present in NMIBC. The majority (63%) was present in low or moderate levels. A restricted group of 18 proteins, 3 of which highly expressed, were exclusively found in MIBC and may play a critical role in advanced disease. In addition, 13% (12/95) of the identified glycoproteins have been previously detected by targeted glycoproteomics for the STn antigen in bladder tumours showing resistance to chemotherapy (identified in red). D ) Target Score for 29 glycoproteins showing some degree of expression in bladder tumours. Briefly, glycoproteins were ranked according to their potential for targeted therapeutics considering its plasma membrane expression in relation to other subcellular locations, absence from healthy urothelium and other human tissues, and overexpression in bladder cancer, while severely penalizing lymphoid system and gametes expression. The overall goal was to maximize tumour specificity while minimizing off-target effects. Maximum possible score was 15 (arbitrary units). GLUT1 (SLC2A1) and HOMER3 were top-ranked proteins scoring 13

Article Snippet: A PierceTM Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific).

Techniques: Glycoproteomics, Mass Spectrometry, Gene Expression, Expressing, Clinical Proteomics, Membrane, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

doi: 10.1186/s13046-021-01988-6

Figure Lengend Snippet: A ) HOMER3 is expressed by the majority of 5637 and T24 cells (> 90%) showing mostly intracellular origin, whereas a small subpopulation also exhibits HOMER3 at the cell surface (3–5%). The left panel shows the expression of HOMER3 at the cell surface in a small subset of T24 cells (3–5%) and a massive expression of total HOMER3 (> 90% of the cells). The graph histogram at right highlights that 5637 and T24 cells presented the same HOMER3 phenotype. Experiments assessing HOMER3 at the cell surface were conducted in cells showing non-permeabilized membranes and without incorporation of propidium iodide. The analysis of permeabilized cells showed HOMER3 total levels. Results are the average of three independent replicates **** p < 0.0001 (student t-test). B ) Immunofluorescence microscopy for HOMER3 (green) in non-permeabilized and permeabilized cells highlighting HOMER3 at the cell membrane (left panel). C ) Immunofluorescence microscopy for HOMER3 (green) at the cell surface of non-permeabilized cells, as confirmed by a uniform expression of CellMask (orange) and the absence of signal for propidium iodine (red). Propidium iodine staining was used as cell death control for exclusion of cells with compromised plasma membranes (lower panel). In addition to PI, dead T24 cells also stained more intensely for cell mask than live cells due to dye diffusion into the intracellular space. D ) Expression of HOMER3 in whole cell extracts, HOMER3 immunoprecipitates (IP) and corresponding supernatants. The blots clearly showed HOMER3 main proteoform at approximately 40 kDa, corresponding to its canonical form. HOMER3 IPs revealed additional bands at 75 and 100 kDa, potentially corresponding to glycoforms, and faint bands bellow 37 kDa. Both cell lines presented similar blot profiles. E ) PNA lectin blots after in situ neuraminidase digestion (NeuAse) for whole cell extracts, HOMER3 immunoprecipitates (IP) and corresponding supernatants. Whole cell extracts show a wide number of bands spanning all molecular weights, consistent with the presence of multiple glycoproteins. Contrastingly, the IP showed bands at 75 and 100 kDa, reinforcing the existence of glycosylation. Moreover, no bands could be observed in the non-glycosylated HOMER3 canonical proteoform. Notably, several signals bellow 37 kDa were observed, suggesting low molecular weight HOMER3 glycoproteoforms. Again, both cell lines presented similar blot profiles. F ) MS/MS spectra for a HOMER3 glycopeptide isolated at 75 kDa highlighting main assignments, peptide fragmentations as well as typical glycans oxonium ions at m/z 204.08 (GalNAc). G ) Estimation of HOMER3 in IP bands by MS for T24 cells showed a wide number of proteoforms of distinct molecular weights (10–200 kDa), with higher abundance between 37 and 75 kDa. Bands were excised from gels, reduced, alkylated, subjected to proteolytic digestion, and analyzed by nanoLC-MS/MS. The presence of HOMER3 in each lane was estimated based on peptide-spectrum match (PSM), i.e. the total number of identified peptide spectra matched for the protein

Article Snippet: A PierceTM Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific).

Techniques: Expressing, Immunofluorescence, Microscopy, Membrane, Staining, Control, Clinical Proteomics, Diffusion-based Assay, In Situ, Glycoproteomics, Molecular Weight, Tandem Mass Spectroscopy, Isolation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

doi: 10.1186/s13046-021-01988-6

Figure Lengend Snippet: Hypoxia and glucose deprivation drive the accumulation of HOMER3 at the cell membrane. A ) Hypoxia and Glucose deprivation significantly increased the number of T24 and 5637 cells expressing HOMER3 at the cell surface. T24 flow cytometry analysis (included in the left panel as a representative example) and graph A (right panel) shows that the vast majority of T24 and 5637 cancer cells express HOMER3, which remains unchanged upon tremendous reduction in oxygen levels and glucose deprivation. On the other hand, this microenvironmental feature promoted a massive increase of cells (approximately 5-fold) showing HOMER3 at the cell surface. B ) Induction of HOMER3 upregulation and downregulation in T24 cells. Blots confirm the development of two HOMER3 knockdown (T24_HOMER3_KD1 and KD2; 30–50% decrease), one knockout model (T24_HOMER3_KO; total HOMER3 abrogation) and an HOMER3 knock-in (T24_HOMER3_KI; 60% increase compared with the control). C ) T24 overexpression is mainly observed at the cell surface after exposure to hypoxia and glucose deprivation. The percentage of cells expressing HOMER3 increases significantly in relation to the controls in both normoxia and hypoxia plus glucose deprivation; however, this increase is more pronounced under microenvironmental pressure. D ) HOMER3 downregulation or deletion leads to a massive decrease in the number of cells expressing the protein in normoxia, irrespectively of the sub-cellular localization, which is not compensated by exposure to hypoxia and glucose deprivation. The results are the average of at least three independent replicates. ** p < 0.01; *** p < 0.001; **** p < 0.0001 (two-way ANOVA with interaction followed by Tukey post hoc)

Article Snippet: A PierceTM Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific).

Techniques: Membrane, Expressing, Flow Cytometry, Knockdown, Knock-Out, Knock-In, Control, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

doi: 10.1186/s13046-021-01988-6

Figure Lengend Snippet: HOMER3 enhances T24 cells proliferation and invasion in normoxia and, particularly, under oxygen shortage and glucose deprivation. T24 wild type cells (T24WT), CRISPR-Cas9 control cells (CTRL), T24_HOMER_KI (with HOMER3 elevation) and KD1–2/KO1 (with HOMER3 downregulation/abrogation) models, all showed decreased proliferation and increased invasion in hypoxia plus glucose deprivation in comparison to normoxia. Notably, T24_HOMER3_KI models were significantly more proliferative and invasive in comparison to wild type and control cells in normoxia and, particularly, under hypoxia and nutrient shortage. The T24_ HOMER3_KD/KO showed the opposite behavior. The results are the average of at least three independent replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (two-way ANOVA with interaction followed by Tukey post hoc)

Article Snippet: A PierceTM Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific).

Techniques: CRISPR, Control, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

doi: 10.1186/s13046-021-01988-6

Figure Lengend Snippet: The HOMER3 membrane phenotype associates with bladder cancer aggressiveness and worst prognosis. A ) HOMER3 is not expressed by the healthy urothelium but could be found at different bladder cancer stages (Ta-T4) and in metastases, with significant accumulation at the cell membrane for high-grade NMIBC and MIBC bladder cancer. HOMER3 was scored based on the number of positive cases and sub-cellular location (cytoplasm vs membrane and cytoplasm). HOMER3 was not expressed in healthy urothelium. On the other hand, the number of positive cases showing membrane staining increased with the severity of the lesions, being also present in most metastases. A significant association between HOMER3 at the cell membrane and high-grade tumours of stages T1 and higher could be observed (Ta vs ≥ T1: 5.3% vs 28.4%; p = 0.038; Chi-square). B ) The overall survival of bladder cancer patients exhibiting HOMER3 at the plasma membrane (memHOMER3) was reduced compared to patients not showing this molecular feature. The survival curve in the upper panel highlights the association between the memHOMER3 and decreased OS (83 vs 148 months; log-rank p = 0.035). The lower panel shows that this is also true for the MIBC subgroup (15 vs 64 months; log-rank p = 0.001)

Article Snippet: A PierceTM Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific).

Techniques: Membrane, Staining, Clinical Proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

doi: 10.1186/s13046-021-01988-6

Figure Lengend Snippet: Sialylated HOMER3 glycoforms can be found at the cell-surface of bladder cancer cells and display significant inter-patient structural variability. A ) HOMER3 at the cell membrane co-localizes in the same tumour area with sialylated Tn and T antigens in bladder cancer. According to immunohistochemistry, HOMER3 was diffusely expressed in wide tumour areas, both at the cytoplasm and cell membranes. These areas co-localized with very high STn and/or ST expressions areas. B ) Immunofluorescence microscopy highlighting the co-localization (in white) of HOMER3 (violet) with sialylated Tn and T antigens (green) at the cell membrane of bladder cancer cells. HOMER3 was detected in both cytoplasm and cell membrane of a high number of cancer cells in invasive tumours. The tumours were also screened for the Tn and T antigens and their sialylated counterparts STn and ST. Tumours were negative for neutral glycans but showed high sialospecies, in accordance with immunohistochemistry analysis in panel A. C ) MS/MS HOMER3 glycopeptide with cancer-associated glycans isolated from an invasive bladder tumour. Briefly, invasive tumours showing no Tn and T antigens were elected for this analysis. Glycoproteins were extracted from these tumours and digested with neuraminidase to expose Tn and T antigens derived from their sialylated counterparts. VVA and PNA lectins were then used to pulldown glycoproteins carrying these glycans for downstream analysis by nanoLC-MS/MS. Peptide and glycopeptide fragmentations as well as typical glycan oxonium ions at m/z 204.09 (GalNAc) and 366.14 (GalNAc-Gal) were used for glycoproteins and glycosites annotations. D ) HOMER3 glycosites mapping by nanoLC-EThcD-MS/MS demonstrated significant inter-patients’ variability. HOMER3 was isolated from five tumour samples of different patients by lectin affinity and characterized in relation to their glycosylation pattern by nanoLC-EThcD-MS/MS. This analysis identified 16 glycosites in the extracellular region of HOMER3 with little homology between different patients. Notably, the identification of glycopeptides carrying the Tn and T antigens strongly supports previous sialylation, since the neutral glycans were not detected in the original tumours

Article Snippet: A PierceTM Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific).

Techniques: Membrane, Immunohistochemistry, Immunofluorescence, Microscopy, Tandem Mass Spectroscopy, Glycoproteomics, Isolation, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

doi: 10.1186/s13046-021-01988-6

Figure Lengend Snippet: HOMER3 and STn are not co-expressed at the cell membrane in relevant human healthy tissues (thyroid, liver, gallbladder, testis, lung, stomach, pancreas, colon, small intestine, appendix). HOMER3 was not detected in these tissues, except for few cells in the submucosa of digestive organs as well as in thyroid follicular cells, all of which showing weak/moderate cytoplasmic staining. HOMER3 was also detected in respiratory tract secretions, suggesting potential translocation of the protein to the extracellular space, warranting confirmation in future studies. Moreover, HOMER3 was not found at the cell surface of healthy cells, contracting with observations for cancer cells. The STn antigens could only be found in mucinous secretions of the gastrointestinal and respiratory tracts, being also scarcely observed at the cell surface of cells facing the lumen of these organs. No evidence of co-localization between both antigens could be observed

Article Snippet: A PierceTM Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific).

Techniques: Membrane, Staining, Translocation Assay