Journal: Frontiers in Immunology

Article Title: Integrative analysis of semaphorins family genes in colorectal cancer: implications for prognosis and immunotherapy

doi: 10.3389/fimmu.2025.1536545

Figure Lengend Snippet: Potential agents for patients with high SRS. (A) Identification of significantly activated pathways in the SRS-high group using the GSEA algorithm. (B) Predicting oxaliplatin sensitivity to assess the computational algorithm’s feasibility. (C, D) Analyzing correlation and differences in drug sensitivity for potential candidates from the CTRP and PRISM datasets. (E–H) Conducting molecular docking studies between SEMA4C and the compounds tanespimycin, clofarabine, nutlin-3, and LGX818.

Article Snippet: Membranes were blocked using 5% skim milk in TBST and incubated with primary antibodies against SEMA4C (1:3000, Cat. no: 28402-1-AP; Proteintech) and β-actin (1:1000, Cat. no: TA09; OriGene Technologies, Inc.).

Techniques:

Journal: Frontiers in Immunology

Article Title: Integrative analysis of semaphorins family genes in colorectal cancer: implications for prognosis and immunotherapy

doi: 10.3389/fimmu.2025.1536545

Figure Lengend Snippet: Verification of the tumor-promoting effect of SEMA4C in CRC. (A) Western blot analysis of SEMA4C expression in HT29 and SW480 cell lines. (B, C) CCK-8 and colony formation assays were conducted to evaluate the impact of SEMA4C downregulation on the proliferation and viability of HT29 and SW480 cell lines. (D–F) Transwell assays were performed to evaluate the influence of SEMA4C downregulation on the cell migration and invasion. (G) Impact of SEMA4C downregulation on the cell apoptosis was analyzed by flow cytometry assay. (*: P < 0.05; **: P < 0.01; ***: P < 0.001).

Article Snippet: Membranes were blocked using 5% skim milk in TBST and incubated with primary antibodies against SEMA4C (1:3000, Cat. no: 28402-1-AP; Proteintech) and β-actin (1:1000, Cat. no: TA09; OriGene Technologies, Inc.).

Techniques: Western Blot, Expressing, CCK-8 Assay, Migration, Flow Cytometry

Journal: Acta biochimica Polonica

Article Title: Circ-POLA2-mediated miR-138-5p/SEMA4C axis affects colon cancer cell activities.

doi: 10.18388/abp.2020_6457

Figure Lengend Snippet: Figure 5. miR-138-5p has a modificatory action on SEMA4C expression (A) Bioinformation sites predicted the binding sites of miR-138-5p and SEMA4C. (B) The binding relationship between miR-138-5p and SEMA4C was verified by dual luciferase reporter assay. (C) SEMA4C in tumor tissues of CC patients was detected by RT-qPCR. (D) The correlation between miR-138-5p and SEMA4C expression in CC tissues was evaluated by Pearson correlation coefficient. (E) SEMA4C in CC cell lines was detected by RT-qPCR. (F) SEMA4C was detected by RT-qPCR and Western blot after miR-138-5p expression intervention; values are expressed as mean ± standard deviation. *P<0.05 vs. sh-NC group; #P<0.05 vs. oe-NC.

Article Snippet: Proteins were separated by 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Beyotime), loaded onto a polyvinylidene fluoride membrane (Millipore), after which 5% nonfat dry milk was added, and primary antibodies SEMA4C (1:10000; sc-136445; Santa Cruz Biotechnology) and GAPDH (1:1000; ab8245; Abcam), and horseradish peroxidaseconjugated secondary antibody (1:5000; sc-2054; Santa Cruz Biotechnology) were supplemented.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Acta biochimica Polonica

Article Title: Circ-POLA2-mediated miR-138-5p/SEMA4C axis affects colon cancer cell activities.

doi: 10.18388/abp.2020_6457

Figure Lengend Snippet: Figure 6. miR-138-5p/SEMA4C axis can intervene the influences of circ-POLA2 on CC (A) The transfection efficacy of sh-POLA2 + anti-miR-138-5p, sh-POLA2 + anti-NC, sh-POLA2 + SEMA4C, or sh-POLA2 + Vector was verified by RT-qPCR and Western blot. (B) Cell viability was detected by CCK-8. (C–D) Cell proliferation was detected by colony formation assay. (E–F) Cell migration and invasion were analyzed by Transwell. (G–H) Apoptosis was analyzed by flow cytometry; values are expressed as mean ± standard deviation. *P<0.05 vs. sh-POLA2+ anti-NC; #P<0.05 vs. sh-POLA2 + Vector.

Article Snippet: Proteins were separated by 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Beyotime), loaded onto a polyvinylidene fluoride membrane (Millipore), after which 5% nonfat dry milk was added, and primary antibodies SEMA4C (1:10000; sc-136445; Santa Cruz Biotechnology) and GAPDH (1:1000; ab8245; Abcam), and horseradish peroxidaseconjugated secondary antibody (1:5000; sc-2054; Santa Cruz Biotechnology) were supplemented.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Colony Assay, Migration, Flow Cytometry, Standard Deviation

Journal: Journal of chemical neuroanatomy

Article Title: Cleavage of semaphorin 4 C interferes with the neuroprotective effect of the semaphorin 4 C/Plexin B2 pathway on experimental intracerebral hemorrhage in rats.

doi: 10.1016/j.jchemneu.2023.102318

Figure Lengend Snippet: Fig. 1. Protein levels of SEMA4C and Plexin B2 in the CNS after ICH. (A) The level of sSEMA4C in the CSF of patients in the non-ICH group (obtained from patients with normal pressure hydrocephalus, n = 10) and the ICH group (obtained from ICH patients within 3 days of onset, n = 16) (Non-ICH vs. ICH: P < 0.001). (B) Pearson correlation coefficient between sSEMA4C levels in the CSF of patients with ICH and GOS score (R = –0.7402, P = 0.001, n = 16). (C) Representative image of brain sections. (D) Western blot analysis and quantification of the time course of the protein level of mSEMA4C (sham vs. ICH 6 h: P = 0.0007, sham vs. ICH 12 h: P = 0.0001, sham vs. ICH 24 h: P < 0.0001, sham vs. ICH 48 h: P = 0.0037, sham vs. ICH 72 h: P = 0.0281, sham vs. ICH 168 h: P = 0.9998) and sSEMA4C (sham vs. ICH 6 h: P < 0.0001, sham vs. ICH 12 h: P < 0.0001, sham vs. ICH 24 h: P = 0.0001, sham vs. ICH 48 h: P < 0.0001, sham vs. ICH 72 h: P = 0.0042, sham vs. ICH 168 h: P = 0.9973) in the brain tissue around the hematoma (n = 6). (E) Western blot analysis and quantification of the time course of the protein level of Plexin B2 in the brain tissue around the hematoma (sham vs. ICH 6 h: P = 0.9980, sham vs. ICH 12 h: P = 0.9958, sham vs. ICH 24 h: P = 0.9453, sham vs. ICH 48 h: P = 0.9503, sham vs. ICH 72 h: P = 0.9871, sham vs. ICH 168 h: P = 0.9506, n = 6). (F) Co-IP analysis and quantification of both mSEMA4C and sSEMA4C interacting with Plexin B2 in the brain tissue around the hematoma (ICH 24 h vs. sham: P < 0.0001, n = 6). In a, d, e and f, the data are presented as the mean ± SD; NS: No significant difference vs. sham group, *P < 0.05 vs. sham group, * *P < 0.01 vs. sham group, * **P < 0.001 vs. sham group.

Article Snippet: Anti-SEMA4C antibody (sc-136445, dilution ratio 1:500 for western blot) and anti-ID-3 antibody (sc-56712, dilution ratio 1:1000 for western blot) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Co-Immunoprecipitation Assay

Journal: Journal of chemical neuroanatomy

Article Title: Cleavage of semaphorin 4 C interferes with the neuroprotective effect of the semaphorin 4 C/Plexin B2 pathway on experimental intracerebral hemorrhage in rats.

doi: 10.1016/j.jchemneu.2023.102318

Figure Lengend Snippet: Fig. 3. Effects of SEMA4C on neuronal death and degeneration in brain tissue. (A) Western blot analysis and quantification of the transduction efficiency of SEMA4C (sham vs. ICH: P < 0.0001, ICH vs. ICH + LV-Con: P = 0.7035, ICH + LV-Con vs. ICH + LV-SEMA4C: P = 0.0146, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.3657) and sSEMA4C protein levels (sham vs. ICH: P ＜ 0.0001, ICH vs. ICH + LV-Con: P = 0.9997, ICH + LV-Con vs. ICH + LV-SEMA4C: P = 0.0082, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P ＜ 0.0001), n = 6. (B) Double staining for TUNEL (green) neuronal marker (NeuN, red). DAPI (blue) represents the nuclei. TUNEL-positive cells are indicated by arrows; scale bar = 100 µm, n = 6. (C) Counts of TUNEL-positive cells (Sham vs. ICH: P < 0.0001, ICH vs. ICH + Vehicle: P > 0.9999, ICH + Vehicle vs. ICH + rSEMA4C: P = 0.8712, ICH vs. ICH + LV-Con: P = 0.9883, ICH + LV-Con vs. ICH + LV-SEMA4C: P < 0.0001, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.8712). (D) FJB staining of brain sections at 24 h after ICH. FJB-positive cells are indicated by arrows; scale bar = 100 µm, n = 6. (E) Counts of FJB-positive cells (sham vs. ICH: P < 0.0001, ICH vs. ICH + Vehicle: P > 0.9999, ICH + Vehicle vs. ICH + rSEMA4C: P = 0.0092, ICH vs. ICH + LV-Con: P > 0.9999, ICH + LV-Con vs. ICH + LV-SEMA4C:P < 0.0029, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.0092). All data are presented as the mean ± SD, in a and c, * **P < 0.01 vs. sham group, #P < 0.05 vs. ICH + LV-Con group, ##P < 0.01 vs. ICH + LV-Con group, in e, * **P < 0.01 vs. sham group, ##P < 0.01 vs. ICH + vehicle group, $$P < 0.01 vs. ICH + LV-Con group.

Article Snippet: Anti-SEMA4C antibody (sc-136445, dilution ratio 1:500 for western blot) and anti-ID-3 antibody (sc-56712, dilution ratio 1:1000 for western blot) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Transduction, Double Staining, TUNEL Assay, Marker, Staining

Journal: Journal of chemical neuroanatomy

Article Title: Cleavage of semaphorin 4 C interferes with the neuroprotective effect of the semaphorin 4 C/Plexin B2 pathway on experimental intracerebral hemorrhage in rats.

doi: 10.1016/j.jchemneu.2023.102318

Figure Lengend Snippet: Fig. 4. Effect of SEMA4C on neurological function after ICH. (A) Adhesive-removal test (sham vs. ICH: P < 0.0001, ICH vs. ICH + Vehicle: P > 0.9999, ICH + Vehicle vs. ICH + rSEMA4C: P = 0.0314, ICH vs. ICH + LV-Con: P > 0.9999, ICH + LV-Con vs. ICH + LV-SEMA4C: P = 0.0269, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.0006, n = 10). (B) Rotarod test (sham vs. ICH: P < 0.0001, ICH vs. ICH + Vehicle: P > 0.9999, ICH + Vehicle vs. ICH + rSEMA4C: P = 0.0330, ICH vs. ICH + LV-Con: P > 0.9999, ICH + LV-Con vs. ICH + LV-SEMA4C: P = 0.0481, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.0446, n = 10). (C-E) Representative images showing swim paths on days 23 (learning) and 26 (memory) after ICH. Escape latency in the Morris water maze test at 21–25 days after ICH (sham vs. ICH: P < 0.0001, ICH vs. ICH + vehicle: P = 0.9878, ICH + vehicle vs. ICH + rSEMA4C: P < 0.0001, ICH vs. ICH + LV-Con: P > 0.9999, ICH + LV-Con vs. ICH + LV- SEMA4C: P < 0.0001, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.0004, n = 10). The time spent in the second quadrant at 26 days after ICH (sham vs. ICH: P = 0.0029, ICH vs. ICH + vehicle: P = 0.9801, ICH + vehicle vs. ICH + rSEMA4C: P = 0.0326, ICH vs. ICH + LV-Con: P > 0.9999, ICH + LV-Con vs. ICH + LV- SEMA4C: P = 0.0402, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.0454, n = 10). Mean swimming speed of each group in the Morris water maze test after ICH (sham vs. ICH: P = 0.9305, ICH vs. ICH + Vehicle: P > 0.9999, ICH + vehicle vs. ICH + rSEMA4C: P > 0.9999, ICH vs. ICH + LV-Con: P > 0.9999, ICH + LV-Con vs. ICH + LV-SEMA4C: P = 0.9997, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.9937, n = 10). All data are presented as the mean ± SD, NS: No significant difference vs. sham group, * *P < 0.01 vs. sham group, * **P < 0.001 vs. sham group, #P < 0.05 vs. ICH + vehicle, ###P < 0.001 vs. ICH + vehicle group, $P < 0.05 vs. ICH + LV-Con group, $$$P < 0.001 vs. ICH + LV-Con group.

Article Snippet: Anti-SEMA4C antibody (sc-136445, dilution ratio 1:500 for western blot) and anti-ID-3 antibody (sc-56712, dilution ratio 1:1000 for western blot) were purchased from Santa Cruz Biotechnology.

Techniques: Adhesive

Journal: Journal of chemical neuroanatomy

Article Title: Cleavage of semaphorin 4 C interferes with the neuroprotective effect of the semaphorin 4 C/Plexin B2 pathway on experimental intracerebral hemorrhage in rats.

doi: 10.1016/j.jchemneu.2023.102318

Figure Lengend Snippet: Fig. 5. Transfection efficiency of SEMA4C overexpression in vitro and its effect on neuronal degeneration. (A) Immunofluorescent staining for flag (green). Nuclei were fluorescently labeled with DAPI (blue). The diagram shows the transfection efficiency (LV-Con-Flag vs. LV-SEMA4C-Flag: P = 0.8330, LV-Con-Flag vs. LV- SEMA4C-secr-Flag: P = 0.9339, n = 3); scale bar = 50 µm. (B) Live/dead staining: green staining indicates viable cells, and red staining indicates dead cells (con trol vs. OxyHb: P < 0.0001, OxyHb vs. OxyHb + LV-Con: P = 0.5393, OxyHb + LV-Con vs. OxyHb + LV-SEMA4C: P = 0.0055, OxyHb + LV-Con vs. OxyHb + LV- SEMA4C-secr: P < 0.0001, n = 3); scale bar = 200 µm. All data are presented as the mean ± SD, NS: No significant difference vs. OxyHb + LV-Con-Flag group, * **P < 0.001 vs. control group, ##P < 0.01 vs. OxyHb + LV-Con group, ###P < 0.001 vs. OxyHb + LV-Con group.

Article Snippet: Anti-SEMA4C antibody (sc-136445, dilution ratio 1:500 for western blot) and anti-ID-3 antibody (sc-56712, dilution ratio 1:1000 for western blot) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Over Expression, In Vitro, Staining, Labeling, Control

Journal: Journal of chemical neuroanatomy

Article Title: Cleavage of semaphorin 4 C interferes with the neuroprotective effect of the semaphorin 4 C/Plexin B2 pathway on experimental intracerebral hemorrhage in rats.

doi: 10.1016/j.jchemneu.2023.102318

Figure Lengend Snippet: Fig. 6. Roles of mSEMA4C and its cleaved form sSEMA4C in brain injury after ICH. Western blot analysis and quantification of ID1 (A, sham vs. ICH: P < 0.0001, ICH vs. ICH + vehicle: P = 0.8791, ICH + Vehicle vs. ICH + rSEMA4C: P = 0.9980, ICH vs. ICH + LV-Con: P = 0.9994, ICH + LV-Con vs. ICH + LV-SEMA4C: P = 0.0033, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P > 0.9999), ID3 (B, sham vs. ICH: P = 0.0492, ICH vs. ICH + vehicle: P = 0.9979, ICH + vehicle vs. ICH + rSEMA4C: P > 0.9999, ICH vs. ICH + LV-Con: P > 0.9999, ICH + LV-Con vs. ICH + LV-SEMA4C: P = 0.0459, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P > 0.9999), and cleaved caspase-3 (C, sham vs. ICH: P < 0.0001, ICH vs. ICH + vehicle: P = 0.9758, ICH + vehicle vs. ICH + rSEMA4C: P = 0.4876, ICH vs. ICH + LV-Con: P = 0.9989, ICH + LV-Con vs. ICH + LV-SEMA4C: P = 0.0183, ICH + LV-Con vs. ICH + LV-SEMA4C-secr: P = 0.0388) (n = 6). (D) Relative RhoA activity was tested by GLISA in vitro (control vs. OxyHb: P = 0.0159, OxyHb vs. OxyHb + LV-Con: P = 0.9973, OxyHb + LV-Con vs. OxyHb + LV-SEMA4C: P = 0.0216, OxyHb + LV-Con vs. OxyHb + LV-SEMA4C-secr: P = 0.0026, n = 3). (E) Western blot analysis and quantification of effects of GM6001 on the protein level of sSEMA4C in brain tissue after ICH (sham vs. ICH: P = 0.0003, ICH vs. ICH + vehicle: P = 0.3518, ICH + vehicle vs. ICH + GM6001: P = 0.0001, n = 6). All data are displayed as means ± SD; in a, b and c, *P < 0.05 vs. sham group, * **P < 0.001 vs. sham group, #P < 0.05 vs. ICH + LV-Con group, ##P < 0.01 vs. ICH + LV-Con group; in d, *P < 0.05 vs. control group, #P < 0.05 vs. OxyHb + LV-Con group, ##P < 0.01 vs. OxyHb + LV-Con group; in e, * **P < 0.001 vs. sham group, ###P < 0.001 vs. ICH + vehicle group.

Article Snippet: Anti-SEMA4C antibody (sc-136445, dilution ratio 1:500 for western blot) and anti-ID-3 antibody (sc-56712, dilution ratio 1:1000 for western blot) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Activity Assay, In Vitro, Control

Journal: Journal of chemical neuroanatomy

Article Title: Cleavage of semaphorin 4 C interferes with the neuroprotective effect of the semaphorin 4 C/Plexin B2 pathway on experimental intracerebral hemorrhage in rats.

doi: 10.1016/j.jchemneu.2023.102318

Figure Lengend Snippet: Fig. 7. Potential mechanisms of SEMA4C and Plexin B2 in brain injury after ICH. After ICH, SEMA4C could interact with Plexin B2, which initiated both forward and reverse signaling. SEMA4C reverse signaling plays a dominant role and shows the “ideal” repair effect. SEMA4C is cleaved into sSEMA4C by the activation of MMPs, which fails to elicit the neuroprotective effect of SEMA4C.

Article Snippet: Anti-SEMA4C antibody (sc-136445, dilution ratio 1:500 for western blot) and anti-ID-3 antibody (sc-56712, dilution ratio 1:1000 for western blot) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Abundance ranking of PB2 and Sema4 family members based on average spectral counts among 398 identified adhesion/surface proteins across 122 treatment-naïve primary breast patient samples ( https://doi.org/10.1016/j.cell.2020.10.036 ); the canonical binding ligands of PB2, Sema4C, Sema4D, and Sema4A, are shown by yellow dots. B ) KM plot of distant metastasis-free survival (DMFS) of breast cancer patients based on mRNA expression of Sema4C, p=0.0051. C ) KM plot of overall survival (OS) of breast cancer patients based on mRNA expression of Sema4C, p=0.049.

Article Snippet: Primary antibodies used include: Anti-PB2 (Protein Tech #10602-1-AP), Anti-Bactin (Abcam #AB8224), Anti-Sema4A (Thermo Fisher #PA5- 101258), Anti-Sema4C (Ray Biotech #102-11819), Anti-Sema4G (Santa Cruz Biotech #sc- 515644), Anti-Sema4D/CD100 (Santa Cruz Biotech #sc-39065), Anti-Sema7C (Santa Cruz Biotech #sc-376149).

Techniques: Binding Assay, Expressing

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Schematic of PB2-Sema4C intercellular interactions in homotypic tumor cell clustering between two cancer cells. B ) Immunoblots of PB2 and Sema4C expression in different breast cancer cell lines and PDX models using antibodies that are specific to detect both human and mouse isoforms. C ) Immunoblots of Sema4C, CDC42, and PB2 in the protein complex immunoprecipitated by anti-PB2 antibody compared to IgG. D ) Immunoblot image with reduced Sema4C after Sema4C KD in MDA-MB-231 tumor cells. E & F) Representative images (E) and average cluster growth area curves (F) of MDA-MB-231 control and PB2 KO cells in co-culture with Con or Sema4C KD cells, measured by IncuCyte, N=3 experiments with at least 5 technical replicates each, PB2 + Sema4C + vs. PB2 + Sema4 - p=1.86e-9, PB2 + Sema4C + vs. PB2 - Sema4C - p=6.81e-10, PB2 - Sema4C + vs. PB2 - Sema4C - p=0.22, PB2 + Sema4C - vs. PB2 - Sema4C - p=0.47. G ) Principal component analysis clusters of MDA-MB-231 single cells, siCon clusters, and siPB2 clusters analyzed from global mass spectrometry with N=3 replicates per group. H ) Heat map of differentially expressed proteins from global mass spectrometry analysis of siCon single cells (blue) N=3, siCon clusters (green) N=3, and siPB2 clusters (red) N=3 I ) Gene ontology biological processes analysis of significantly up-regulated and down-regulated proteins in PB2 control vs. PB2 KD clusters in breast cancer cells analyzed by mass spectrometry. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: Primary antibodies used include: Anti-PB2 (Protein Tech #10602-1-AP), Anti-Bactin (Abcam #AB8224), Anti-Sema4A (Thermo Fisher #PA5- 101258), Anti-Sema4C (Ray Biotech #102-11819), Anti-Sema4G (Santa Cruz Biotech #sc- 515644), Anti-Sema4D/CD100 (Santa Cruz Biotech #sc-39065), Anti-Sema7C (Santa Cruz Biotech #sc-376149).

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Co-Culture Assay, Mass Spectrometry