Fig 1: Overexpression of RORA reduces LPS-induced renal epithelial cell apoptosis. qRT-PCR (A) and Western blotting (B) were used to detect the expression of RORA mRNA and protein in HK-2 cells treated with different concentrations of LPS (0, 1, 2, 5 and 10 µg/mL). qRT-PCR (C) and Western blotting (D) were used for analyzing the transfection efficiency of OE-RORA in LPS-treated HK-2 cells. E CCK-8 was used for evaluating the proliferation of LPS-treated HK-2 cells before and after OE-RORA transfection. ELISA was performed to detect the expression of IL-6 (F) and IL-1ß (G) in the supernatant of LPS-treated HK-2 cells before and after OE-RORA transfection. H ELISA was performed to detect the activity of caspase-3 in LPS-treated HK-2 cells before and after OE-RORA transfection. I Western blotting was used to detect caspase-3 and cleaved caspase-3 in LPS-treated HK-2 cells before and after OE-RORA transfection. J Flow cytometry was applied for analyzing the apoptosis of LPS-treated HK-2 cells before and after OE-RORA transfection. Data were expressed as mean ± standard deviation and each experiment was repeated 3 times. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with 0 µg/mL or control group. ##P < 0.01 and ###P < 0.001, compared with LPS + OE-NC group. Abbreviations: RORA, retinoic acid receptor-related orphan receptor alpha; LPS, lipopolysaccharide; qRT-PCR, quantitative real-time polymerase chain reaction; OE-RORA, RORA overexpression vector; OE-NC, negative control vector; CCK-8, cell counting kit-8; ELISA, enzyme-linked immunosorbent assay; IL, interleukin
Fig 2: Hsa_circ_0000144 depletion suppressed OXA resistance, proliferation and metastasis in OXA-resistant GC cells. (A) CCK-8 assay for the survival rate of AGS, AGS/OXA, MKN45 and MKN45/OXA cells treated with OXA at different concentrations (5, 10, 20, 40, 80, 160 or 320 µM). (B) RT-qPCR assay for the expression of hsa_circ_0000144 in AGS/OXA and MKN45/OXA cells transfected with si-NC, si-hsa_circ_0000144#1, si-hsa_circ_0000144#2 or si-hsa_circ_0000144#3. (C–H) AGS/OXA and MKN45/OXA cells were transfected with si-NC or si-hsa_circ_0000144#1. (C) CCK-8 assay for the survival rate of transfected cells treated with OXA at different concentrations (5, 10, 20, 40, 80, 160 or 320 µM). (D) Colony formation assay for the colony formation ability of transfected cells. (E) Flow cytometry for the cell apoptosis in transfected cells. (F) Caspase 3 activity in transfected cells determined by commercial kit. (G and H) Transwell assay for the cell migration and invasion in transfected cells. *P < 0.05.
Fig 3: CK treatment leads to the generation of ROS in renal cell carcinoma cells. (A) Intracellular ROS levels were measured by flow cytometry. (B) Cell viability was measured via an MTT assay. (C) Cell migration was measured by a wound healing assay. (D) Cell invasion was measured via a Transwell invasion assay. (E) Cellular apoptosis was measured. (F) The indicated proteins were measured via western blotting. (G) Caspase-3 activity was analysed. The data are presented as the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control group or as indicated. CK, ginsenoside compound K; ROS, reactive oxygen species.
Fig 4: RORA alleviates LPS-induced renal epithelial cell apoptosis by promoting PGC-1a transcription. LPS-treated HK-2 cells were transfected with OE-RORA + sh-PGC-1a or OE-RORA + sh-NC. qRT-PCR (A) and Western blotting (B) were used to detect PGC-1a mRNA and protein. C CCK-8 was used for evaluating cell proliferation. ELISA was performed to detect the expression of IL-6 and IL-1ß (D) and the activity of caspase-3 (E). F Western blotting was used to detect caspase-3 and cleaved caspase-3. G Flow cytometry was applied for analyzing cell apoptosis. Data were expressed as mean ± standard deviation and each experiment was repeated 3 times. #P < 0.05, ##P < 0.01 and ###P < 0.001, compared with OE-NC + sh-NC group. &P < 0.05, &&P < 0.01 and &&&P < 0.001, compared with OE-RORA + sh-NC group. RORA, retinoic acid receptor-related orphan receptor alpha; PGC-1a, peroxisome proliferator-activated receptor gamma coactivator 1 alpha; LPS, lipopolysaccharide; OE-RORA, RORA overexpression vector; sh-PGC-1a, PGC-1a inhibition vector; sh-NC, negative control vector; qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, cell counting kit-8; ELISA, enzyme-linked immunosorbent assay; IL, interleukin
Fig 5: Overexpression of miR-93 suppresses LPS-induced renal cell apoptosis. (A) TCMK-1 cells were treated with different concentrations of LPS (0, 0.01, 0.1, 1 and 10 µg/ml) for 24 h, and the expression of miR-93 was detected by RT-qPCR. (B) The transfection efficiency of miR-93 mimics was detected by RT-qPCR. (C) miR-93 mimics were added to the cultured TCMK-1 cells for 24 h, and treated with LPS, and then cell viability was assessed by Cell Counting Kit-8 assay. (D) Activity of Caspase-3 was measured using a Caspase-3 activity assay kit. (E) Apoptosis was detected by flow cytometry. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. Control group; ##P<0.01 vs. LPS group. miR, microRNA; LPS, lipopolysaccharide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control.
Supplier Page from Abcam for Caspase-3 Activity Assay Kit (Fluorometric)