Fig 1.
BMH-21 impacts RPA12 nucleolar localization.
(A) Immunofluorescence staining of A375 melanoma cells treated with BMH-21 for the indicated times. Cells were stained for RPA12 and fibrillarin (FBL). DNA was counterstained using Hoechst. Representative biological replicate of n = 3 is shown. Arrowheads indicate nucleolar cap structures. Scale bar, 10 μm. (B) Quantification of the images for RPA12. Fold change is shown. N = 50 cells per treatment. (C, D) Ectopic expression of RPA12-DDK. RPA12-DDK expression vector was transfected into A375 cells at the indicated amounts (μg) and treated with BMH-21 (1 μM) for three hours. Cell lysates (30 μg) were analyzed with antibodies for RPA194, DDK and RPA12. (D) Quantification of RPA12-DDK in (C). N = 2–3 replicates. Mean fold change ±SD is shown. Student’s two-tailed t-test **, p = 0.00545; ns, non-significant. (E) qPCR analysis of RPA12 transcript in A375 melanoma cells treated with BMH-21 for 3 hours. Mean fold change ±SD of n = 3 biological replicates is shown. Statistical analysis was conducted using Student’s two-tailed t test. ns, non-significant.
Fig 2.
RPA12 knockdown affects the expression and localization of RNA Polymerase I subunits RPA194 and RPA135.
(A) qPCR analysis of RPA12 transcript in A375 melanoma cells with transient RPA12 knockdown using siRNA. Mean fold change ± SD of n = 4 biological replicates is shown. Mann-Whitney two-tailed t-test *, p < 0.05. (B) Immunofluorescence staining of RPA12. Representative biological replicate of n = 4 is shown. (C) Quantification of the images for RPA12. Mean fold change ±SD of n = 3 biological replicates is shown. Analysis of N = 50 cells per treatment. Student’s two-tailed t-test **, p = 0.0018. (D) Immunofluorescence staining of siCtrl and siRPA12 knockdown cells. A375 melanoma cells were stained for the indicated Pol I subunits and fibrillarin (FBL) following siRNA knockdown of RPA12. DNA was counterstained using Hoechst. Representative biological replicates of n = 4 are shown. (E) Quantification of the images for RPA194. Mean fold change ±SD of n = 3 biological replicates is shown. N = 50–100 cells per treatment. Student’s two-tailed t-test *, p = 0.0306. (F) Immunofluorescence staining of siCtrl and siRPA12 knockdown cells for PAF53 and CAST. (G) Quantification of the images for PAF53 and CAST. N = over 100 cells per treatment. Scale bars, 10 μm.
Fig 3.
Depletion of RPA12 does not modify the effect of BMH-21 on RPA194.
(A) Immunofluorescence staining of siCtrl and RPA12 knockdown cells. A375 melanoma cells were stained for RPA194 and FBL following siRNA knockdown of RPA12. DNA was counterstained using Hoechst. Cells were treated with vehicle (Ctrl) or BMH-21 (1 μM) for 3 hours. Representative biological replicate of n = 4 is shown. Scale bar, 10 μm. (B) Quantification of the images for RPA194. Fold change is shown. N = 2 biological replicates and 50–150 cells per treatment.
Fig 4.
Influence of RPA12 on RPA194 and RPA135 proteins and their interaction.
Ctrl siRNA and RPA12 siRNA transfected A375 melanoma cells were treated with BMH-21 (1 μM) for 3 hours. (A) Western blot analysis. Cell lysates (20 μg/lane) were immunoblotted for RPA194 and RPA135. (B) Quantification for RPA194 of n = 5 biological replicates and RPA135 of n = 3 biological replicates. Data are represented as mean ± SD. Statistical analysis was conducted using non-parametric Mann-Whitney two-tailed t test. ns, non-significant. *, p<0.05. (C and D) Coimmunoprecipitation analyses. Cell lysates (C) were immunoprecipitated with RPA194 and RPA135 antibodies or control IgG followed by immunoblotting (D) for RPA194 and RPA135 as indicated. Molecular weight markers are shown to the left.
Fig 5.
RPA12 is not essential for Pol I transcription, nor the enzyme chromatin occupancy.
(A) qPCR analysis of rRNA synthesis following transfection with siCtrl and RPA12 siRNAs in A375 melanoma cells. Cells were treated with and without BMH-21 (1 μM) for 6 hours. Primer pairs for 5’ETS and 18S rRNAs were used. Fold change for n = 3 biological replicates are shown, and data are represented as mean ± SD. Statistical analysis was conducted using non-parametric Mann-Whitney two-tailed test. ns, non-significant. (B) Schematic outline for the rRNA coding region and the probes used for Northern analysis (blue) and primers used for ChIP (red). (C and D) Northern blot analyses. (C) A375 cells were treated with BMH-21 for the indicated times and total RNA was prepared. 6 μg RNA/lane was loaded. (D) Total RNA was isolated from A375 melanoma cells transfected with siCtrl, RPA12 or RPA135 siRNAs and treated with or without BMH-21 for 6 hours. RNA (0.9 μg per lane) was loaded and the blots were probed with ITS1 and 28S probes as indicated. rRNA processing transcripts are indicated to the left. (E) Chromatin immunoprecipitation analysis following siCtrl and RPA12 knockdown in A375 melanoma cells. Primer pairs for the promoter (-48), 5’ETS (+851), 18S (+4446), two termination sites (+13508, +15364) and non-coding IGS (+30541) regions were used. Fold enrichment of n = 4 biological replicates is shown. Data are represented as mean ± SD. Statistical analysis was conducted using non-parametric Mann-Whitney two-tailed test. ns, non-significant.
Fig 6.
Deletion of RPA12 renders yeast cells partially resistant to BMH-21.
Ten-fold serial dilutions of WT, rpa12.2Δ, and dst1Δ strains were spotted onto YEPD plates with indicated concentrations of BMH-21. Plates were incubated for six days at 23°C.