Fig 1: Regulation of the expression of rDNA genes and ribosomal protein genes by hnRNP UL1. (A) Scheme showing the 47S pre-rRNA with the positions of the primers and probes used in qPCR and northern blotting, respectively. The sites of PCR primer pairs are indicated in blue: A - on rDNA promoter, B - on 47S rDNA, C - on 45S rDNA, D - on 18S rDNA, E - on 5.8S rDNA, F - on 28S rDNA, and G - on IGS rDNA. Yellow indicates probes for northern blotting: 5'ETS, ITS1 and ITS2. (B) RNA Pol I binding at different regions of the rDNA loci (promoter, IGS, 5.8S, 18S, 28S, 45S, and 47S) was quantified by ChIP combined with qPCR in HEK UL1 KO cells compared to HEK WT cells. 5S rRNA was used as a negative control. (C) Total RNA extracted from HEK WT and HEK UL1 KO cells was separated on a denaturing gel and analyzed by northern blotting. The blots were probed with the oligonucleotides 5'ETS, ITS1 and ITS2. The detected pre-rRNA species are highlighted. (D) The mRNA levels of two ribosomal proteins (RPS6 and RPS15) and the nucleolar marker fibrillarin (FIB) were analyzed by RT-qPCR in HEK UL1 KO cells compared to HEK WT cells. The error bars represent the SDs of three biological replicates. P values were calculated using Student's t test, and the statistical significance is represented as follows: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig 2: RPS6 knock down promotes cell cycle G1 phase arrest in ovarian cancer. SKOV3 and HO-8910 cell cycles were analyzed by flow cytometry. Representative images showing cell cycle arrest in G0G1 phase on RPS6 depletion are presented (a). Histogram illustrating the percentage of cells in each cell cycle phase. The results reveal that the proportion of cells in G0G1 phase increased, while the proportion in G2M phase decreased in SKOV3 and HO-8910 cells on RPS6 knockdown (b and c). Levels of cyclin D1, cyclin E, CDK2, CDK4, CDK6, and pRb were analyzed by western blotting following RPS6 knockdown in both SKOV3 and HO-8910 cells (d and e). All comparisons are with the LV-shCon control group; ***p < 0.001
Fig 3: Localization of organelle markers in ECIs of RCCs. Immunohistochemical staining showed that ECIs were intensively positive for PEX14 and CAT1 (two markers of peroxisomes, arrows in A and B). Some of the ECIs were positive for GM130 (a marker of Golgi apparatus, arrows in C). None of the ECIs exhibited immunoreactivity of LAMP1 and LAMP2 (two markers of lysosome or autolysosome, arrowheads in D and E), TOM20 (a marker of mitochondria, arrowheads in F), CALR (a marker of the endoplasmic reticulum, arrowheads in G), RPS6 (a marker of ribosomes, arrowheads in H), or RAB5A (a marker of early endosomes, arrowheads in I). Double- immunofluorescence labelling of ECIs revealed that most of the NBR1-positive ECIs were positive for PEX14 and CAT1, and that a portion of them displayed moderate GM130 staining (J).
Fig 4: Knockdown of RPS6 inhibited ovarian cancer cell migration and invasion in vitro. Migration ability was assessed using the scratch test after 24 and 48 h (magnification 40×; scale bar: 500 μm) on SKOV3 (a, b) and HO-8910 (c, d) cells. Transwell migration assays were conducted to assess the effect of RPS6 knockdown on SKOV3 and HO-8910 cell migration (magnification 100×; scale bar: 200 μm) (e). Graph of transwell migration assay data for SKOV3 cells with RPS6 knocked down (112 ± 4.90 and 122 ± 8.38), showing reduced cell migration, compared with control cells (shCon) (350 ± 12.25) (N = 3, 2-tailed unpaired t-test, p < 0.0001) (f). Graph of transwell migration assay data for HO-8910 cells with RPS6 knocked down (47 ± 5.73 and 63 ± 5.25), showing reduced cell migration, compared with shCon (288 ± 4.64) (N = 3, 2-tailed unpaired t-test, p < 0.0001) (g). Representative images showing Matrigel invasion assays using SKOV3 and HO-8910 cells with RPS6 knocked down and respective controls (magnification, 200×; scale bar: 200 μm) (h). Representative graph of Matrigel invasion assay data for SKOV3 cells with RPS6 knocked down (15.33 ± 0.47 and 24.8 ± 4.95), compared with controls (shCon) (176 ± 20.05) (N = 3, 2-tailed unpaired t-test, p < 0.001) (i). Representative graph of Matrigel invasion assay data for HO-8910 cells with RPS6 knocked down (26.7 ± 3.30 and 19.0 ± 7.93) compared with controls (shCon) (149.1 ± 22.70) (N = 3, 2-tailed unpaired t-test, p < 0.001) (J). Data are presented as mean ± SD for three independent experiments; ***p < 0.001, ****p < 0.0001
Fig 5: PYCARD-AS1 suppresses PYCARD translation.(A) The indicated biotin-labelled transcripts were used to pull down GAPDH or PYCARD mRNA from SKBR3 cell lysates. The retrieved mRNA was subjected to qRT-PCR analysis, and the data of retrieved mRNA are relative to the values for the control beads. The used PYCARD-AS1ΔOS transcript is shown schematically in (C, upper). (B) MS2-RIP assay detecting the PYCARD mRNA associated with PYCARD-AS1 or PYCARD-AS1ΔOS. GAPDH mRNA was detected as a negative RNA control. (C) RNase-A assay detecting the association between endogenous PYCARD-AS1 and PYCARD transcripts. RNA extracted from the RNase A-treated SKBR3 cell lysates was subjected to qRT-PCR analysis with primer sets that span the non-overlapping and overlapping regions (1 and 2, shown schematically), or with primers detecting GAPDH mRNA. (D) Schematic representation of the distribution of RNA, ribosomal subunits, ribosomes and polysomes along fractions of an increasing sucrose gradient (top to bottom fractions). RNA and protein abundances were determined by measuring the absorbance at 254 and 280 nm (upper); RNA samples extracted from gradient fractions were visualized with ethidium bromide (lower). (E) PYCARD mRNA (upper), but not GAPDH mRNA (lower), was increased in heavy polysomes by PYCARD-AS1 knockdown. The relative distribution of PYCARD or GAPDH mRNA was determined by qRT-PCR analysis of RNA in gradient fractions, and was presented as a percentage of the total RNA in the gradient. Data represent mean values from three independent experiments. (F) Native RIP detecting PYCARD (upper) or GAPDH (lower) mRNA retrieved by L26- or RPS6-specific antibody in SKBR3 cells with or without PYCARD-AS1 knockdown. (G, H) qRT-PCR (G) and immunoblotting (H) detecting the PYCARD expression level in response to PYCARD-AS1 knockdown with or without complementation with PYCARD-AS1OS or PYCARD-AS1ΔOS. In (A, B, C, F and G), data are presented as means ± SD from three independent experiments performed in triplicate; *p < 0.05; **p < 0.01; ***p < 0.001.
Supplier Page from Abcam for Anti-RPS6 antibody - Ribosome Marker