Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a Representative images of immunohistochemistry (IHC) for B7-H3 and CD31 in CRC tissues and matched normal tissues from the 125 clinical CRC patients. Scale bar, 100 μm. b , c B7-H3 ( b ) and CD31 ( c ) protein expression based on their staining index in CRC specimens and matched normal tissues. d CD31 protein expression is shown in patients stratified into B7-H3 low (median value) groups. e Correlation analysis of the staining index of the protein expression levels of B7-H3 and CD31 in human CRC specimens ( n = 125). The correlation coefficient (r) is shown. The data represent the means ± SEM. *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a Western blot analysis of B7-H3 in CRC stable cell lines with B7-H3 inhibition (shB7-H3) or their control cell lines (sh-NC) (left). Western blot analysis of B7-H3 in CRC stable cell lines overexpressing B7-H3 (B7-H3) or their control cell lines (EV) (right). β-actin served as a loading control. b , c Cell migration ( b ) and invasion ( c ) in HUVECs were examined by transwell assays after HUVECs were plated and treated with conditioned medium (CM) from sh-NC cells or shB7-H3 cells. Scale bar, 100 μm. One representative image from three reproducible experiments is shown. Migrated and invaded HUVEC numbers are shown in the bar graph. d Effect of CM from sh-NC cells or shB7-H3 cells on tube formation in HUVECs. Scale bar, 100 μm. The number of tubes counted is shown in the bar graph. One representative result from three reproducible experiments is shown. e , f Cell migration ( e ) and invasion ( f ) in HUVECs were examined by transwell assays after HUVECs were plated and treated with CM from EV cells or B7-H3 cells. Migrated and invaded HUVEC numbers are shown in the bar graph. g Effect of CM from EV cells or B7-H3 cells on tube formation in HUVECs. The number of tubes counted is shown in the bar graph. The data represent the means ± SEM. ** P < 0.01, *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Western Blot, Stable Transfection, Inhibition, Migration

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a The expression of angiogenesis-related genes was detected by RT-qPCR in shB7-H3 HCT116 and RKO cells. b Western blot analysis of B7-H3 and VEGFA in the sh-NC and shB7-H3 CRC cell lines. β-actin served as a loading control. c Representative images of IHC for VEGFA in CRC tissues and matched normal tissues from the 125 clinical CRC patients. Scale bar, 100 μm. d VEGFA protein expression based on the staining index of CRC specimens and matched normal tissues. e VEGFA protein expression is shown for patients stratified into B7-H3 low (median value) groups. f Correlation analysis of the staining index of the protein expression levels of B7-H3 and CD31 in human CRC specimens ( n = 125). The correlation coefficient (r) is shown. The data represent the means ± SEM. NS no significant difference; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a , b Cell migration ( a ) and invasion ( b ) in HUVECs were examined by transwell assays after HUVECs were co-treated with CM from sh-NC cells or shB7-H3 cells and IgG or recombinant VEGFA (rVEGFA). Migrated and invaded HUVEC numbers are shown in the bar graph. c The tube formation of HUVECs co-treated with CM from sh-NC cells or shB7-H3 cells and IgG or rVEGFA was examined. The number of tubes counted is shown in the bar graph. d , e Cell migration ( d ) and invasion ( e ) in HUVECs were examined by transwell assays after HUVECs were co-treated with CM from EV cells or B7-H3 cells and siRNA negative control (NC) or VEGFA siRNA. Migrated and invaded HUVEC numbers are shown in the bar graph. f The tube formation of HUVECs co-treated with CM from EV cells or B7-H3 cells and NC or VEGFA siRNA was examined. The number of tubes counted is shown in the bar graph. The data represent the means ± SEM. NS no significant difference; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Migration, Recombinant, Negative Control

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a The expression of p-STAT3, p-p65, p-AKT, p-JNK, and p-ERK1/2 in HCT116 (left) and RKO (right) stable cell lines with B7-H3 inhibition (shB7-H3) or their control cell lines (sh-NC) was analyzed by Western blot. β-actin was used as an internal control. b Relative mRNA level of VEGFA in B7-H3 CRC cells after treatment with perifosine, BAY11–7082 or cryptotanshinone were analyzed by RT-qPCR. c The protein expression of VEGFA in B7-H3 CRC cells after treatment with perifosine, BAY11–7082 or cryptotanshinone was analyzed by ELISA. d The protein expression of VEGFA in B7-H3 CRC cells after treatment with BAY11–7082 was detected by Western blot. e NF-κB activity in EV cells or B7-H3 cells was examined by luciferase reporter assay. f Schematic representation of the proposed B7-H3/NF-κB/VEGFA axis. g , h Cell migration ( g ) and invasion ( h ) in HUVECs were examined by transwell assays after HUVECs were co-treated with CM from EV cells or B7-H3 cells and BAY11–7082. Migrated and invaded HUVEC numbers are shown in the bar graph. i The tube formation of HUVECs co-treated with CM from EV cells or B7-H3 cells and BAY11–7082 was examined. The number of tubes counted is shown in the bar graph. The data represent the means ± SEM. NS no significant difference; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Expressing, Stable Transfection, Inhibition, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Luciferase, Reporter Assay, Migration

Journal: Cell Death & Disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: a , b CD31 ( a ) and VEGFA ( b ) protein expression based on their IHC staining index results in subcutaneous tumors formed by sh-NC-HCT116 and shB7-H3-HCT116 cells. N = 5. c , d CD31 ( c ) and VEGFA ( d ) protein expression based on their IHC staining index results in subcutaneous tumors formed by EV-HCT116 and B7-H3-HCT116 cells. N = 5. e , f CD31 ( e ) and VEGFA ( f ) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with BAY11–7082 (6 mg/kg). g , h CD31 ( g ) and VEGFA ( h ) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with bevacizumab (1 mg/kg). i , j CD31 ( i ) and VEGFA ( j ) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors co-treated with 3E8 (5 mg/kg) and BAY11–7082 (6 mg/kg) or bevacizumab (1 mg/kg). N = 5. The data represent the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: To demonstrate the effect of soluble B7-H3 on VEGFA expression, cell culture supernatants of CRC cells treated with or without 50 ng/ml recombinant human B7-H3 (rB7-H3, Sino Biological, Beijing, China, #11188-H02H) for 24 h were collected.

Techniques: Expressing, Immunohistochemistry

Journal: Cancer Medicine

Article Title: Higher preoperative serum levels of PD ‐L1 and B7‐H4 are associated with invasive and metastatic potential and predictable for poor response to VEGF ‐targeted therapy and unfavorable prognosis of renal cell carcinoma

doi: 10.1002/cam4.754

Figure Lengend Snippet: Cox regression analysis for various potential prognostic factors in overall survival in all cases

Article Snippet: The serum levels of B7 family molecules, CD28, and VEGF were measured by using the following ELISA kits: human CD80 (CSB‐E15768h, Cusabio Biotech, Wuhan, China), human CD86 (CSB‐E08542h, Cusabio Biotech, Wuhan, China), human B7‐H4 (CSB‐E15013h, Cusabio Biotech, Wuhan, China), human PD‐1 (CSB‐E13643h, Cusabio Biotech, Wuhan, China), human PD‐L1 (CSB‐E13644h, Cusabio Biotech, Wuhan, China), human CD28 (CSB‐E09296h, Cusabio Biotech, Wuhan, China), human B7‐H3 (DB7H30, R&D Systems, Minneapolis, MN), human VEGF (DVE00, R&D Systems Minneapolis, MN), and human CTLA‐4 (KA0165, Abnova, Taipe, Taiwan), as described previously .

Techniques:

Journal: Cancer Medicine

Article Title: Higher preoperative serum levels of PD ‐L1 and B7‐H4 are associated with invasive and metastatic potential and predictable for poor response to VEGF ‐targeted therapy and unfavorable prognosis of renal cell carcinoma

doi: 10.1002/cam4.754

Figure Lengend Snippet: Cox regression analysis for various potential prognostic factors in recurrence‐free survival in 108 N0M0 cases at radical nephrectomy

Article Snippet: The serum levels of B7 family molecules, CD28, and VEGF were measured by using the following ELISA kits: human CD80 (CSB‐E15768h, Cusabio Biotech, Wuhan, China), human CD86 (CSB‐E08542h, Cusabio Biotech, Wuhan, China), human B7‐H4 (CSB‐E15013h, Cusabio Biotech, Wuhan, China), human PD‐1 (CSB‐E13643h, Cusabio Biotech, Wuhan, China), human PD‐L1 (CSB‐E13644h, Cusabio Biotech, Wuhan, China), human CD28 (CSB‐E09296h, Cusabio Biotech, Wuhan, China), human B7‐H3 (DB7H30, R&D Systems, Minneapolis, MN), human VEGF (DVE00, R&D Systems Minneapolis, MN), and human CTLA‐4 (KA0165, Abnova, Taipe, Taiwan), as described previously .

Techniques:

Journal: JACS Au

Article Title: Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

doi: 10.1021/jacsau.2c00328

Figure Lengend Snippet: (A,B) Structural and functional design of a LTzC . The construct comprises: (a) BMS-202-derived end (blue) to target PD-L1-overexpressing cells; (b) PEG-linker (black), as a solubility tag; (c) a tetrazine (magenta) for IEDDA reactions; and (d) dithiolane ring (green), as a redox agent. BMS-202 attachment strategy was identified using molecular modelling studies (PDB: 5J89 ). (C) Synthesis of LTzC . (a) Pd(OAc) 2 , Xphos, CsCO 3 , toluene, 80 °C, 24 h, 47%. (b) tert -Butyl (2-(ethylamino)ethyl)carbamate, sodium triacetoxyborohydride, DCE, room temperature, 24 h, 53%. (c) (i) 1 N HCl in dioxane, 2 h; (ii) Amberlyst A-21, DCM, 30 min; (iii) BocNH-PEG6-COOH, BOP, DIPEA, DMF, r.t., overnight, 88%. (d) (i) 1 N HCl in dioxane, 2 h; (ii) Amberlyst A-21, DCM, 30 min; (iii) Tz 1 , BOP, DIPEA, r.t., overnight, 62%. (e) (i) 1 N HCl in dioxane, 2 h; (ii) lipoic acid, BOP, DIPEA, DMF, r.t., overnight, LTzC = 47%. Overall yield = 6.4%. (D) Synthesis of Non-targeting LTzC . (f) (i) TFA/DCM (1:1), 30 min; (ii) lipoic acid, BOP, DIPEA, DMF, r.t., overnight, 59%. (g) Boc(NH)-PEG7-NH 2 , BOP, DIPEA, DMF, r.t., overnight, 42%. Overall yield = 24.7%.

Article Snippet: The membrane was blocked with a blocking buffer for 1 h at r.t., followed by incubation with the primary antibody (1:1000, Human PD-L1 Mab, R&D Systems Inc.) at 4 °C for 24 h. The membrane was washed twice with TBST, followed by incubation with the secondary antibody (1:1000, Goat Anti-Rabbit IgG HRP, R&D Systems Inc.) for 2 h. The membrane was washed thrice with TBST, followed by addition of Clarity ECL western blotting substrates (Bio-Rad) before imaging in the ChemiDoc XRS + Imaging System (Bio-Rad).

Techniques: Functional Assay, Construct, Derivative Assay, Solubility

Journal: JACS Au

Article Title: Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

doi: 10.1021/jacsau.2c00328

Figure Lengend Snippet: Schematic strategy and confocal imaging of LTzC / SRB probe bioconjugation method. (A) Structure of the fluorescent exo -norbornene-tagged sulforhodamine B probe ( SRB probe ) and ligation strategy with LTzC . (B) Western blot analysis of PD-L1 expression in MDA-MB-231 and MCF-7 in the presence and absence of IFN-γ (activator of PD-L1 expression). (C) Targeted bioorthogonal labeling of PD-L1 proteins in PD-L1-positive and -negative cell lines. Cells were first treated with LTzC (3 μM) for 4 h to enable targeted binding of PD-L1, followed by removal of unbound LTzC and addition of SRB Probe (100 nM) for 24 h. Unbound SRB probe was removed, and the cells were fixed for staining and imaging experiments. Nuclei were stained by Hoechst 33342; actin was stained using Phalloidin-Alexa Fluor 647. PD-L1 was labeled using antibody-labeling (Human PD-L1 Mab/Alexa Fluor 488 Goat anti-Rabbit IgG) post-fixation to study co-localization of its fluorescent signals with bioorthogonal-labeling ( LTzC / SRB Probe ) in MDA-MD-231 cells (merge).

Article Snippet: The membrane was blocked with a blocking buffer for 1 h at r.t., followed by incubation with the primary antibody (1:1000, Human PD-L1 Mab, R&D Systems Inc.) at 4 °C for 24 h. The membrane was washed twice with TBST, followed by incubation with the secondary antibody (1:1000, Goat Anti-Rabbit IgG HRP, R&D Systems Inc.) for 2 h. The membrane was washed thrice with TBST, followed by addition of Clarity ECL western blotting substrates (Bio-Rad) before imaging in the ChemiDoc XRS + Imaging System (Bio-Rad).

Techniques: Imaging, Ligation, Western Blot, Expressing, Labeling, Binding Assay, Staining, Antibody Labeling

Journal: JACS Au

Article Title: Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

doi: 10.1021/jacsau.2c00328

Figure Lengend Snippet: PD-L1 cell surface and total protein expression after IFN-gamma, BMS-202, or LTzC treatment in MDB-MB-231 cell line. (A) Workflow of flow cytometry measurement of surface PD-L1 using antibody-labeling. (B) Quantification of surface PD-L1 via flow cytometry. Data are represented as mean fluorescent intensity ratio of control cells vs treated cells ( n = 3). (C) Western Blot analysis of total PD-L1 expression ( n = 3). Representative western blots are provided for each data set.

Article Snippet: The membrane was blocked with a blocking buffer for 1 h at r.t., followed by incubation with the primary antibody (1:1000, Human PD-L1 Mab, R&D Systems Inc.) at 4 °C for 24 h. The membrane was washed twice with TBST, followed by incubation with the secondary antibody (1:1000, Goat Anti-Rabbit IgG HRP, R&D Systems Inc.) for 2 h. The membrane was washed thrice with TBST, followed by addition of Clarity ECL western blotting substrates (Bio-Rad) before imaging in the ChemiDoc XRS + Imaging System (Bio-Rad).

Techniques: Expressing, Flow Cytometry, Antibody Labeling, Western Blot

Journal: JACS Au

Article Title: Targeted Molecular Construct for Bioorthogonal Theranostics of PD-L1-Expressing Cancer Cells

doi: 10.1021/jacsau.2c00328

Figure Lengend Snippet: (A) Structure and bioorthogonal activation of 7-oxa-norbornadiene-masked prodrugs Pro-INK128 and Pro-Dox . (B) Track-&-treat strategy: PD-L1 positive cells vs PD-L1 negative cells. (C) Dose response curves of INK128 and Pro-INK128 (left) and doxorubicin and Pro-Dox (right) in MDA-MB-231 cells. (D) Study of the LTzC -triggered activation of prodrugs in MDA-MB-231 and MCF-7 cancer cells. Cells were first treated with LTzC (3 μM) for 4 h, followed by removal of unbound LTzC and addition of Pro-Dox (0.3 μM) and/or Pro-INK128 (0.1 μM). Studies were performed with and without IFN-γ treatment to compare different levels of PD-L1 expression. Cell viability was measured at day 5 using PrestoBlue reagent. Negative controls: LTzC (3 μM)/ Pro-Dox (0.3 μM) or Pro-INK128 (0.1 μM); positive control: doxorubicin (0.3 μM) or INK128 (0.1 μM). The data are average of triplicates and the error bars indicate standard deviations.

Article Snippet: The membrane was blocked with a blocking buffer for 1 h at r.t., followed by incubation with the primary antibody (1:1000, Human PD-L1 Mab, R&D Systems Inc.) at 4 °C for 24 h. The membrane was washed twice with TBST, followed by incubation with the secondary antibody (1:1000, Goat Anti-Rabbit IgG HRP, R&D Systems Inc.) for 2 h. The membrane was washed thrice with TBST, followed by addition of Clarity ECL western blotting substrates (Bio-Rad) before imaging in the ChemiDoc XRS + Imaging System (Bio-Rad).

Techniques: Activation Assay, Expressing, Positive Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: GOLM1 is correlated with B7-H3 expression in human ovarian cancer. (a) The mRNA expression of GOLM1 and B7-H3 was analyzed with the Pearson Correlation analysis GEPIA database |Log2FC| cutoff = 1; q -value cutoff = 0.01; ANOVA differential analysis in 426 ovarian cancer (OV) samples and 88 normal ovarian tissue. (b) The GOLM1 and B7-H3 mRNA levels were retrieved from OV dataset ( n = 379) with ENCORI database, R = 0.421. (c) IHC plot of the typical protein expression of GOLM1 and B7-H3 in two OV patients (ID: 1884 and 2347) from The Human Protein Atlas. (d) Statistical analysis of GOLM1 and B7-H3 protein expression in 12 patients from patients with OV from The Human Protein Atlas. (e) Kaplan–Meier plot of the overall survival of 1402 ovarian cancer patients in TCGA database. The logrank p value was 0.0016.

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: B7-H3 expression is partially regulated by GOLM1. (a) qPCR analysis of GOLM1 knockdown efficiency in SKOV3 cell line after stable introduction of three different shRNAs by lentivirus system. (b) qPCR analysis of B7-H3 mRNA expression in three GOLM1 shRNA stable induced SKOV3 cell lines. (c) Western blot analysis of GOLM1 and B7-H3 protein expression in three GOLM1 knockdown SKOV3 cell lines. (d) Statistics of GOLM1 and B7-H3 protein expression in three GOLM1 knockdown SKOV3 cell lines. (e) qPCR analysis of GOLM1 and B7-H3 expression in SKOV3 cell line with transient exogenous expressed GOLM1 using PEI proreagent. (f) Western blot analysis of GOLM1 and B7-H3 expression in GOLM1 overexpressed SKOV3 cell line. (g) Statistics of GOLM1 and B7-H3 expression in GOLM1 overexpressed SKOV3 cell line. Each experiment was repeated three times and values are presented as means ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-tailed Student's t -test).

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: Expressing, shRNA, Western Blot, Two Tailed Test

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: GOLM1 expression was correlated with B7-H3 membrane protein expression and B7-H3 secretion. (a) FACS analysis of comparable B7-H3 membrane expression in GOLM1 knockdown and GOLM1 overexpressed SKOV3 cell lines. Original SKOV3 cell line as the control. (b) Statistics of B7-H3 membrane expression in three SKOV3 cell lines indicated in (a). (c) ELISA assay of soluble B7-H3 expression in three SKOV3 cell lines after cultured in incubator for three indicated times (24 hours, 48 hours, and 72 hours). Three independent experiments are performed, and values are presented as means ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-tailed Student's t -test). (d) The interaction of exogenous expressed GOLM1 and B7-H3 was detected in 293T cells cotransfected with His-GOLM1 and Flag-B7-H3. (e) The interaction endogenously expressed GOLM1, and B7-H3 was detected in SKOV3 cells.

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: GOLM1 regulates ovarian cancer metastasis and invasion ability in soluble B7-H3-dependent manner. (a) Western blot of B7-H3, GOLM1 and metastasis-related protein E-cadherin, MMP-9, and VEGF in GOLM1 knockdown and GOLM1 overexpressed SKOV3 cell lines. Both scramble shRNA knockdown SKOV3 cell line and original SKOV3 cell line are treated as the control. (b) Western blot of lentivirus induced stable knockdown B7-H3 in SKOV3 cell lines compared with original and GOLM1 knockdown SKOV3 cell lines. (c) The Transwell assay to determine the invasiveness of GOLM1 knockdown and B7-H3 knockdown SKOV3 cell lines treated with or without additional soluble B7-H3 (10 μ g/ml) for 48 hours. (d) Statistics of the invaded cell number between each group ( ∗ p < 0.05, ∗∗ p < 0.01). (e) The wound-healing ability of GOLM1 knockdown and B7-H3 knockdown SKOV3 cell lines treated with or without soluble B7-H3 (10 μ g/ml) for 48 hours. (f) Statistics of the percentage of wound area remaining between each group ( ∗ p < 0.05).

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: Western Blot, shRNA, Transwell Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: GOLM1 regulates ovarian cancer progression in vivo. (a) In vivo bioluminescence imaging of 107 GOLM1 and B7-H3 knockdown SKOV3 cell lines subcutaneous injected behind the right upper limb in immunodeficient NSG mice, using original SKOV3 as the control. Each group contains three mice. The imaging was performed at day 0 and day 42. (b) Statistics of BLI signal between three groups ( ∗ p < 0.05, ∗∗ p < 0.01). (c) Vernier caliper measurement of tumor volume every 7 days after tumor inoculation. Statistical difference was calculated at day 42 ( ∗ p < 0.05, ∗∗ p < 0.011). (d) ELISA assay of soluble B7-H3 in peripheral blood of each mouse at day 42 ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (e) In vivo bioluminescence imaging of 107 GOLM1 and B7-H3 knockdown SKOV3 cell lines' tail vein injected in immunodeficient NSG mice, using original SKOV3 as the control. Each group contains three mice. The imaging was performed at day 1 and day 42. (f) Statistics of BLI signal between the three groups ( ∗ p < 0.05). (g) ELISA assay of soluble B7-H3 in peripheral blood of each mice at day 42 ( ∗∗ p < 0.01). (h) Survival curve of the mice in the three groups.

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: In Vivo, Imaging, Injection, Enzyme-linked Immunosorbent Assay