Journal: bioRxiv

Article Title: Targeting Semaphorin 7a signaling in preclinical models of estrogen receptor-positive breast cancer

doi: 10.1101/2025.05.21.655360

Figure Lengend Snippet: A) Gene Ontology (GO) Pathway Analysis of RPPA results, showing top pathways upregulated in MCF7 SEMA7A OE cell lysates, as fold change compared to EV, p<0.05. B) Log2(fold change) of integrin and AKT-related proteins in MCF7 SEMA7A OE versus EV lysates from RPPA-1 data. Multiple two-tailed unpaired t-tests. C) Immunoblot for Integrin β4, Integrin β1 and SEMA7A in elutions following CoIP of MCF7 EV and SEMA7A OE lysates for IgG or SEMA7A. ii. Quantification of IP shown as fold change compared to EV. Two-tailed unpaired t-test. D) Immunoblot for Integrin β4, Integrin β1 and SEMA7A in elutions following CoIP of MCF7 lysates for IgG or SEMA7A, after treatment with PBS (-) or 50 μM RGDS (+). Quantification of IP shown as fold change compared to control. Two-tailed unpaired t-test. Error bars are mean +/-SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Membranes were blocked with 5% bovine serum albumin and probed with the following primary antibodies according to manufacturer recommendation: SEMA7A (Santa Cruz; Cat#Sc-374432), pAKT S473 (Cell Signaling; Cat# 4060), total AKT (Cell Signaling; Cat#2920), Integrin β4 (Cell Signaling; Cat#14803), Integrin β1 (Cell Signaling; Cat#34971), ERα (AbCam; Cat#16660), α-tubulin (Cell Signaling; Cat +#9099S) and GAPDH (Biolegend; Cat# 607902).

Techniques: Two Tailed Test, Western Blot, Control

Journal: bioRxiv

Article Title: Targeting Semaphorin 7a signaling in preclinical models of estrogen receptor-positive breast cancer

doi: 10.1101/2025.05.21.655360

Figure Lengend Snippet: A) Brightfield cell count measured by BioSpa Live Imaging over 96 hours of MCF7 EV and SEMA7A OE cells treated with mouse IgG1 or 1 mM SmAb H1. Two-tailed unpaired t-test at time = 96 hours. B) Representative crystal violet staining of TC11 shCtrl and shSEMA7A KD#2 cells after treatment for 48 hours with mouse IgG1 or 1 mM SmAb H1. Bar graph: quantification of all replicates. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. C) Average tumor volume in TC11 tumor-bearing mice over time, with groups receiving IgG1 or SmAb H1 (n=5 per group). Two-tailed unpaired t-test at day 4, 6, and 8 between (shCtrl) IgG and (shCtrl) SmAb H1 groups.. D) Tumor volume calculated using necropsy tumor photos in TC11 shCtrl and shSEMA7A-tumor bearing mice over time with groups receiving IgG1 or SmAb H1 (n=10 per group). Ordinary one-way ANOVA with Tukey’s multiple comparisons test. E) Weight of the tumor (and attached mammary gland) at study endpoint (n=10/group). Ordinary one-way ANOVA with Tukey’s multiple comparisons test. F) Immunohistochemistry staining of ex vivo TC11 tumors from IgG-treated and SmAb H1-treated groups. Scale bars = 50mm. Hematoxylin and eosin (H&E) staining (i-ii), active integrin B1 (9EG7) staining; brown (iii-iv), integrin B4 staining; brown (v-vi), and SEMA7A staining; brown (vii-viii). G) Log2(fold change) of pAKT (S473) expression in SmAb H1-treated versus IgG-treated TC11 tumors from RPPA-2 data. Two-tailed unpaired t-test. H) Quantification of 9EG7 (active ITGB1) IHC staining from all tumors (n=5 per group); Percent positive out of tumor area. Two-tailed unpaired t-test. I) Quantification of ITGB4 IHC staining from all tumors (n=5 per group); Percent positive out of tumor area. Two-tailed unpaired t-test. J) Quantification of SEMA7A IHC staining from all tumors (n=5 per group); percent positive out of tumor area. Two-tailed unpaired t-test. Error bars are mean +/- SD (A-B), or mean +/- SEM (C-E, G-I). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Membranes were blocked with 5% bovine serum albumin and probed with the following primary antibodies according to manufacturer recommendation: SEMA7A (Santa Cruz; Cat#Sc-374432), pAKT S473 (Cell Signaling; Cat# 4060), total AKT (Cell Signaling; Cat#2920), Integrin β4 (Cell Signaling; Cat#14803), Integrin β1 (Cell Signaling; Cat#34971), ERα (AbCam; Cat#16660), α-tubulin (Cell Signaling; Cat +#9099S) and GAPDH (Biolegend; Cat# 607902).

Techniques: Cell Counting, Imaging, Two Tailed Test, Staining, Immunohistochemistry, Ex Vivo, Expressing