Journal: bioRxiv

Article Title: Collagen Prolyl Hydroxylases Regulate HIF-α Levels Independently of pVHL in ccRCC

doi: 10.1101/2025.09.14.676157

Figure Lengend Snippet: A) Western blot analysis showing the effect of knockdown of P4HA1 (P1), P4HA2 (P2), and P4HB (P4B) or non-targeting control (Con) siRNA on HIF-1α, HIF-2α, and phosphorylated S6 (S235/236) (P-S6) in ACHN cells (VHL wild-type, VHLwt) in normoxia or hypoxia for the last 16 hours prior to lysis. B) Western blot analysis showing the combined effect of knockdown of P4HA1 and P4HA2 (P1/2) using siRNAs from Dharmacon and Ambion, on HIF-1α, HIF-2α, and P-S6 in RCC4 cells expressing mutant (VHL mut) or wild-type pVHL in normoxia or hypoxia for the last 24 hours prior to lysis. C) Western blots showing the combined effect of P4HA1 and P4HA2 knockdown on HIF-1α, HIF-2α, and P-S6 in three VHL-mutant ccRCC cell lines, UMRC2, 786-0, and UOK101 in normoxia. D) Western blot analysis of RCC4 and RCC10 cells treated with the collagen prolyl-4-hydroxylase inhibitor diethyl-pythiDC (dp-DC) for 48 hours under normoxic or hypoxic (4hrs) conditions. E, F) Western blot analysis of RCC4 cells (pVHL mutant) under normoxic conditions, (E), and ACHN cells (pVHL wild-type) exposed to hypoxia for 20 hours (F), showing the effects of P4HA1 (P1) and P4HA2 (P2) overexpression on P-S6, P-ERK, HIF-1α, and HIF-2α levels. G) Western blot analysis of RCC4 cells (pVHL mutant) treated with collagen 1 (Col 1) for 24 hours under normoxic conditions after transfection with control (Con) siRNA or siRNA targeting P4HA1 and P4HA2 (P1/2). Solid lines indicate demarcation of blots from separate gels.

Article Snippet: Renal cell carcinoma cell lines ACHN and 786-0 were purchased from ATCC (Manassas, VA), whereas UOK101 and RCC10 were generous gifts from M. Celeste Simon (University of Pennsylvania).

Techniques: Western Blot, Knockdown, Control, Lysis, Expressing, Mutagenesis, Over Expression, Transfection

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