Journal: Gland Surgery

Article Title: A comprehensive pan-cancer analysis revealing SPAG6 as a novel diagnostic, prognostic and immunological biomarker in tumor

doi: 10.21037/gs-24-157

Figure Lengend Snippet: Expression of SPAG6 in the clinical data. (A) The differential expression of SPAG6 in pan-cancer (red) and normal tissues (blue) downloaded from different databases. (B) The correlation between tumor stage and SPAG6 expression. Stage I = blue, stage II = red, stage III = green, and stage IV = purple. (C) The correlation between patients’ gender and SPAG6 expression. Male = red and female = blue. (D) The correlation between patients’ grade and SPAG6 expression. Grade 1 = green, grade 2 = blue, grade 3 = red, and grade 4 = purple. (E) The correlation between patients’ age and SPAG6 expression. –, no statistical significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. G, grade.

Article Snippet: The sections were then incubated overnight with a primary antibody against spag6 (anti-SPAG6, 1:200, ab155653, Abcam, Cambridge, MA, USA) at 4 °C.

Techniques: Expressing

Journal: Gland Surgery

Article Title: A comprehensive pan-cancer analysis revealing SPAG6 as a novel diagnostic, prognostic and immunological biomarker in tumor

doi: 10.21037/gs-24-157

Figure Lengend Snippet: Prognostic value of SPAG6 across cancers. (A) The correlation between OS and SPAG6 expression. (B-F) Kaplan-Meier analysis of significant outcomes in OS, namely DLBC, LAML, ALL, PAAD, and TGCT. (G) The correlation between DSS and SPAG6 expression. (H) The correlation between DFI and SPAG6 expression. (I) The correlation between PFI and SPAG6 expression. HR, hazard ratio; CI, confidence interval; NaN, not a number; L, low; H, high; OS, overall survival; DLBC, lymphoid neoplasm diffuse large b-cell lymphoma; LAML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; PAAD, pancreatic adenocarcinoma; TGCT, testicular germ cell tumor; DSS, disease-specific survival; DFI, disease-free interval; PFI, progression-free interval.

Article Snippet: The sections were then incubated overnight with a primary antibody against spag6 (anti-SPAG6, 1:200, ab155653, Abcam, Cambridge, MA, USA) at 4 °C.

Techniques: Expressing

Journal: Gland Surgery

Article Title: A comprehensive pan-cancer analysis revealing SPAG6 as a novel diagnostic, prognostic and immunological biomarker in tumor

doi: 10.21037/gs-24-157

Figure Lengend Snippet: The expression of SPAG6 was associated with heterogeneity, gene mutation, and RNA modifications. (A-C) The lollipop plots indicate that SPAG6 was associated with genetic heterogeneity, namely TMB (A), MSI (B), and tumor ploidy (C). (D) The correlation between SPAG6 and gene mutation. Wild type = red and mutation = blue. (E) The landscape of the single-nucleotide variants of SPAG6 . (F) The correlation between RNA modifications and SPAG6 expression. –, no statistical significance; *, P<0.05. WT, wild type; Mut, mutant; TMB, tumor mutational burden; MSI, microsatellite instability.

Article Snippet: The sections were then incubated overnight with a primary antibody against spag6 (anti-SPAG6, 1:200, ab155653, Abcam, Cambridge, MA, USA) at 4 °C.

Techniques: Expressing, Mutagenesis

Journal: Gland Surgery

Article Title: A comprehensive pan-cancer analysis revealing SPAG6 as a novel diagnostic, prognostic and immunological biomarker in tumor

doi: 10.21037/gs-24-157

Figure Lengend Snippet: The association between SPAG6 and tumor-related immunity in the tumor microenvironment. The correlation between tumor purity and SPAG6 expression. (A-D) The correlation between SPAG6 and infiltration as calculated by ESTIMATE score. (E-G) The correlation between SPAG6 and infiltration as calculated by immune score. (H-J) The correlation between SPAG6 and infiltration as calculated by stromal score. (K) The correlation between immune-related cells and SPAG6 expression. (L) The correlation between immunomodulators and SPAG6 expression. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. DC, dendritic cell; MHC, major histocompatibility complex.

Article Snippet: The sections were then incubated overnight with a primary antibody against spag6 (anti-SPAG6, 1:200, ab155653, Abcam, Cambridge, MA, USA) at 4 °C.

Techniques: Expressing

Journal: Gland Surgery

Article Title: A comprehensive pan-cancer analysis revealing SPAG6 as a novel diagnostic, prognostic and immunological biomarker in tumor

doi: 10.21037/gs-24-157

Figure Lengend Snippet: Correlation of SPAG6 expression as tumor stemness and immune checkpoint genes and the enrichment analysis its interacting targets. (A,B) The association between SPAG6 expression and tumor stemness, including DNAss (A) and RNAss (B). (C) The association between SPAG6 expression and immune checkpoints in pan-cancer. (D) The protein-protein interaction network of SPAG6 . (E) The Gene Ontology enrichment analysis of SPAG6 and its interacting targets. *, P<0.05. GO, Gene Ontology.

Article Snippet: The sections were then incubated overnight with a primary antibody against spag6 (anti-SPAG6, 1:200, ab155653, Abcam, Cambridge, MA, USA) at 4 °C.

Techniques: Expressing

Journal: Gland Surgery

Article Title: A comprehensive pan-cancer analysis revealing SPAG6 as a novel diagnostic, prognostic and immunological biomarker in tumor

doi: 10.21037/gs-24-157

Figure Lengend Snippet: Experimental validation of SPAG6 expression. (A) Multiple immunofluorescence staining of SPAG6 in pan-cancer tissue sections. Views of cancers, including high expression (LIHC, CHOL, and OV) and low expression (UCEC, CESC, and THCA) under microscopy (200×). SPAG6 = red, cellular nuclei = blue (DAPI). (B) Cell Counting Kit 8 assay of SPAG6 . (C) Transwell migration assay of SPAG6 (10× magnification). The cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Migrated cells were photographed with a microscope. ***, P<0.001. LIHC, liver hepatocellular carcinoma; CHOL, cholangiocarcinoma; OV, ovarian serous cystadenocarcinoma; UCEC, uterine corpus endometrial carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; THCA, thyroid carcinoma; DAPI, 4,6-diamidino-2-phenyiindole 2 hci.

Article Snippet: The sections were then incubated overnight with a primary antibody against spag6 (anti-SPAG6, 1:200, ab155653, Abcam, Cambridge, MA, USA) at 4 °C.

Techniques: Expressing, Immunofluorescence, Staining, Microscopy, Cell Counting, Transwell Migration Assay

Journal: Gland Surgery

Article Title: A comprehensive pan-cancer analysis revealing SPAG6 as a novel diagnostic, prognostic and immunological biomarker in tumor

doi: 10.21037/gs-24-157

Figure Lengend Snippet: The effect of SPAG6 overexpression on the THCA cell lines. (A) Differential expression of SPAG6 in normal and tumor tissues. (B,C) Prognostic value of SPAG6 in THCA. (D) Mutated genes in high and low SPAG6 expression group. (E-H) GSEA of SPAG6 . –, no statistical significance; *, P<0.05; ****, P<0.0001. THCA, thyroid carcinoma; HR, hazard ratio; CI, confidence interval; GSEA, gene set enrichment analysis.

Article Snippet: The sections were then incubated overnight with a primary antibody against spag6 (anti-SPAG6, 1:200, ab155653, Abcam, Cambridge, MA, USA) at 4 °C.

Techniques: Over Expression, Expressing

Journal: iScience

Article Title: Identification of IQCH as a calmodulin-associated protein required for sperm motility in humans

doi: 10.1016/j.isci.2023.107354

Figure Lengend Snippet: Association of IQCH with calmodulin in flagella and its requirement for sperm capacitation (A) Western blot analysis of protein tyrosine phosphorylation following in vitro capacitation of spermatozoa from the patient and a control individual, at 37°C for 3 h. The data presented for the control individual are representative of the profiles obtained with semen samples from five control individuals displaying normal semen parameters following WHO criteria ( Figure S3 A). (B) Assessment of the IQCH/calmodulin interaction by proximity ligation assay (PLA). Analysis of the physical proximity of IQCH and calmodulin (CaM) relative to the biological negative control (DNAI2-SPAG6, two proteins from different axonemal complexes) and positive control (DNAI2-DNAH17, two proteins from the same axonemal complex). (C) 3D structures of IQ motifs bound to calmodulin in the calcium free status. Side chains of the residues involved in IQ-CaM interactions are shown as sticks and colored blue or orange for polar and hydrophobic contacts, respectively. Structural and sequence alignments of the IQ motifs from IQCH, IQCG, and Myosin V are shown.

Article Snippet: In addition to the experiment, a number of experimental controls were performed: (I) a technical negative control without primary antibody; (II) a biological negative control with non-interacting proteins, such as DNAI2 (mouse monoclonal, Abnova H00064446-M01, 1/320 dilution) and SPAG6 (rabbit polyclonal, Sigma HPA038440, 1/200 dilution); (III) a biological positive control with interacting proteins, such as DNAI2 (mouse monoclonal, Abnova H00064446-M01, 1/320) and DNAH17 (rabbit polyclonal Sigma HPA024354, 1/320 dilution); and (IV) the testing condition with IQCH (rabbit polyclonal, Sigma HPA040845 antibody; 1/100 dilution) and calmodulin (mouse monoclonal, Upstate 05-173; 1/100 dilution).

Techniques: Western Blot, In Vitro, Proximity Ligation Assay, Negative Control, Positive Control, Sequencing

Journal: iScience

Article Title: Identification of IQCH as a calmodulin-associated protein required for sperm motility in humans

doi: 10.1016/j.isci.2023.107354

Figure Lengend Snippet:

Article Snippet: In addition to the experiment, a number of experimental controls were performed: (I) a technical negative control without primary antibody; (II) a biological negative control with non-interacting proteins, such as DNAI2 (mouse monoclonal, Abnova H00064446-M01, 1/320 dilution) and SPAG6 (rabbit polyclonal, Sigma HPA038440, 1/200 dilution); (III) a biological positive control with interacting proteins, such as DNAI2 (mouse monoclonal, Abnova H00064446-M01, 1/320) and DNAH17 (rabbit polyclonal Sigma HPA024354, 1/320 dilution); and (IV) the testing condition with IQCH (rabbit polyclonal, Sigma HPA040845 antibody; 1/100 dilution) and calmodulin (mouse monoclonal, Upstate 05-173; 1/100 dilution).

Techniques: Recombinant, Protease Inhibitor, Electron Microscopy, Plasmid Preparation, Gel Extraction, SYBR Green Assay, Imaging, In Situ, Software