achn human cell line  (European Collection of Authenticated Cell Cultures)

Journal: Journal of Inflammation Research

Article Title: Sirp-α Antibody Inhibits Renal Cell Carcinoma Progression via Akt1/Akt2 Modulation in Tumor-Associated Macrophages

doi: 10.2147/JIR.S530775

Figure Lengend Snippet: The Sirp-α mAb inhibited RCC-induced M2 macrophage polarization by regulation of the PI3K/AKT pathway. ( A ) The STRING mutual drawing of the Akt1 and Akt2 with the 25 up-regulated intersection genes. ( B ) The STRING mutual drawing of Sirp-α with PI3K, Akt1 and Akt2. ( C ) The co-cultivation mode diagram to test the effect of the tumor cells on the M2 macrophage polarization (the THP-1 cells induced with PMA and IL-4) in RCC cell line (ACHN)-conditioned medium. ( D ) Representative contour plots from flow cytometry analysis of the frequencies of the CD206 + /CD86 + macrophages. ( E ) The macrophages killing assay to detect the killing ability of macrophages against tumors in a co-cultured system of the macrophages and the ACHN cells with different conditions. ACHN+M (the ACHN and macrophages co-cultured groups), ACHN+M+Sirp-α (Sirp-α mAb was added at a concentration of 10 μg/mL to the macrophages cells), CD47 − ACHN+M (the macrophages were co-cultured with the CM of the ACHN cells knockdown CD47), CD47 − ACHN+M+Sirp-α (the macrophages disposed with the Sirp-α mAb then were co-cultured with the CM of the ACHN cells knockdown CD47). ( F and G ) The protein expression of PI3K, p-PI3K, Akt1, Akt2 was detected using Western blotting and densitometric analysis. ( H and I ) The protein expression of M2 macrophage marker (CD163, Arg-1, and IL-10) expression in the four groups using Western blotting and densitometric analysis. ( J ) The qRT-PCR analysis of M1 macrophage marker (IL-1, IL-6, IL-12, and TNF-α) expression of the four groups. ( K ) The ELISA analysis of M1 macrophage marker (IL-1, IL-6, IL-12, and TNF-α) expression of the four groups. M2 groups (the isolate M2 cells groups), M2 + PI3K groups (the PI3K overexpressed groups: Lenti-PI3K was added at the beginning of the stimulation), M2 + Sirp-α groups (Sirp-α mAb was added at a concentration of 10 μg/mL to the cells) and M2 + PI3K + Sirp-α groups (Sirp-α mAb was added at a concentration of 10 μg/mL to the cells after transferred Lenti-PI3K). Data are representative of three independent experiments. Values are shown as means±SD from three independent experiments. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The human RCC cell lines (ACHN) and mice renal cell lines (Renca) from the American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Biological Industries, USA), penicillin (100 U/mL), and streptomycin (100 g/mL).

Techniques: Flow Cytometry, Cell Culture, Concentration Assay, Knockdown, Expressing, Western Blot, Marker, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: Sirp-α Antibody Inhibits Renal Cell Carcinoma Progression via Akt1/Akt2 Modulation in Tumor-Associated Macrophages

doi: 10.2147/JIR.S530775

Figure Lengend Snippet: The Sirp-α mAb inhibited RCC cell migration and invasion i n vivo . ( A ) The Co-cultivation mode diagram to test the effect of the M2 macrophage on the ACHN cells. ( B ) The scratch wound healing assays in ACHN cells (scale bar=100 μm). ( C and D ) Transwell assay to assess migration and invasion capacity of the ACHN cells treated with or without Sirp-α mAb and the statistical analysis (scale bar=100 μm). ( E and F ) Western blots and grey value of EMT-related protein expression in ACHN cells. ACHN + M2 groups (ACHN cells were exposed with the isolate M2 cells groups), ACHN + M2 + PI3K groups (Lenti-PI3K was added to the M2 at the beginning of the stimulation), ACHN + M2 + Sirp-α groups (Sirp-α mAb was added at a concentration of 10 μg/mL to the cells) and ACHN + M2 + PI3K + Sirp-α groups (Sirp-α mAb was added at a concentration of 10 μg/mL to the cells after transferred Lenti-PI3K). Data are representative of three independent experiments. ( G ) Immunofluorescence of Ki-67 (red fluorescence) in the mice tumor tissues. (scale bar: 20 μm). ( H ) Bioluminescence imaging of mice after cell injection into the tail vein. ACHN cells were co-injected with macrophages subjected to the indicated treatment. Metastasis was monitored and imaged by bioluminescence using an in vivo imaging system. M2 groups (the isolate M2 cells groups), M2 + PI3K groups (the PI3K overexpressed groups: Lenti-PI3K was added at the beginning of the stimulation), M2 + Sirp-α groups (Sirp-α mAb was added at a concentration of 10 μg/mL to the cells) and M2 + PI3K + Sirp-α groups (Sirp-α mAb was added at a concentration of 10 μg/mL to the cells after transferred Lenti-PI3K). ( I ) Immunofluorescence of Ki-67 (red fluorescence) in the mice tumor tissues. (scale bar: 20 μm). ( J ) Bioluminescence imaging of mice after cell injection into the tail vein. Metastasis was monitored and imaged by bioluminescence using an in vivo imaging system. Representative images are shown from a total of 3 animals per group. Values are shown as means±SD from three independent experiments. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The human RCC cell lines (ACHN) and mice renal cell lines (Renca) from the American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Biological Industries, USA), penicillin (100 U/mL), and streptomycin (100 g/mL).

Techniques: Migration, Transwell Assay, Western Blot, Expressing, Concentration Assay, Immunofluorescence, Fluorescence, Imaging, Injection, In Vivo Imaging

Journal: Biomedicines

Article Title: Immunomodulatory Effects of High-Dose Irradiation Regimens in Renal Cell Carcinoma: Insights from an In Vitro Model with Human Peripheral Blood Mononuclear Cell

doi: 10.3390/biomedicines13092107

Figure Lengend Snippet: Impact of high dose irradiation on viability and proliferation of RCC cell lines, ( A ) cells viability, and ( B ) cells proliferation. The data are presented as normalized on control condition (0 Gy); results obtained 24 h after irradiation. Two-way ANOVA test was performed, Tukey’s post hoc test was applied to correct for multiple comparisons with p ≤ 0.05 (*), p ≤ 0.001 (***) and p ≤ 0.0001 (****) ( n = 6 per dose group). ( C ) Analysis of mortality types in ACHN and CAKI-2 cell lines, 24 h post-irradiation using Annexin V-FITC/PI staining assay. At the right Annexin V/IP FACS plot in both cell lines. Histogram presentation at the left with the values expressed as a percentage of the parent population, where the values were compared with the non-treated. Two-way ANOVA test was performed ( n = 5), with p ≤ 0.05 (*), p ≤ 0.001 (***), and p ≤ 0.0001 (****) being considered statistically significant or highly significant.

Article Snippet: Two human renal cell carcinoma (RCC) lines were used: ACHN (ATCC CRL-1611, Manassas, VA, USA), originally isolated from the pleural effusion of a 22-year-old male with metastatic renal adenocarcinoma, and Caki-2 (ATCC HTB-47, Manassas, VA, USA), derived from a primary clear cell carcinoma of a 69-year-old Caucasian male.

Techniques: Irradiation, Control, Staining

Journal: Biomedicines

Article Title: Immunomodulatory Effects of High-Dose Irradiation Regimens in Renal Cell Carcinoma: Insights from an In Vitro Model with Human Peripheral Blood Mononuclear Cell

doi: 10.3390/biomedicines13092107

Figure Lengend Snippet: Differential expression of PD-L1, VEGF, HIF-1α, and inflammation-associated cytokine genes 24 h post-irradiation in both RCC cell lines, comparing irradiated versus non-irradiated controls: ( A ) ACHN cell line and ( B ) CAKI-2 cell line. Histograms show the 2 −ΔΔCT expression levels of the genes of interest. Data represent four independent experiments performed in triplicate, expressed as means ± SEM ( n = 4) and normalized to each cell line’s untreated control condition (0 Gy). Statistical differences were analyzed using a non-parametric Mann–Whitney multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Two human renal cell carcinoma (RCC) lines were used: ACHN (ATCC CRL-1611, Manassas, VA, USA), originally isolated from the pleural effusion of a 22-year-old male with metastatic renal adenocarcinoma, and Caki-2 (ATCC HTB-47, Manassas, VA, USA), derived from a primary clear cell carcinoma of a 69-year-old Caucasian male.

Techniques: Quantitative Proteomics, Irradiation, Expressing, Control, MANN-WHITNEY, Comparison

Journal: Biomedicines

Article Title: Immunomodulatory Effects of High-Dose Irradiation Regimens in Renal Cell Carcinoma: Insights from an In Vitro Model with Human Peripheral Blood Mononuclear Cell

doi: 10.3390/biomedicines13092107

Figure Lengend Snippet: Quantification of pro-inflammatory and immunosuppressive cytokine secretion by activated PBMCs cultured with conditioned media from irradiated or non-irradiated RCC cell lines. Cytokine concentrations were measured by multiplex ELISA in the supernatants of activated peripheral blood mononuclear cells (PBMC-A) from healthy donors cultured for 72 h with conditioned media from ACHN and Caki-2 cells, collected 24 h after exposure to irradiation or control conditions. Data are normalized to cytokine levels in activated PBMCs cultured with tumor conditioned media not treated (PBMC-A—sn NT) and are presented as mean ± SEM. Results represent three independent experiments. ( A ): Pro-inflammatory Cytokine secretion following PBMC-A culture with ACHN irradiated or not supernatants and Caki-2 irradiated or not supernatants. ( B ): Immunosuppressive Cytokine secretion following PBMC-A culture with ACHN irradiated or not supernatants and Caki-2 irradiated or not supernatants. Results are presented as means ± SEM ( n = 5). Statistical differences between conditions were assessed using a One-way ANOVA test, Tukey’s post hoc test was applied to correct for multiple comparisons, with significance levels set at p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****), where p ≤ 0.05 is considered statistically significant and the others highly significant. (sn = supernatants).

Article Snippet: Two human renal cell carcinoma (RCC) lines were used: ACHN (ATCC CRL-1611, Manassas, VA, USA), originally isolated from the pleural effusion of a 22-year-old male with metastatic renal adenocarcinoma, and Caki-2 (ATCC HTB-47, Manassas, VA, USA), derived from a primary clear cell carcinoma of a 69-year-old Caucasian male.

Techniques: Cell Culture, Irradiation, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Control