Figure 2: CINAP deletion inhibits 18S rRNA processing and blocks rapid cell growth.
From: The ATPase hCINAP regulates 18S rRNA processing and is essential for embryogenesis and tumour growth
(a) Schematic diagram of rRNA processing in eukaryotes. The 5′-ITS1 and 5′-ITS2 probes used in the northern blot assay are indicated. (b) Total RNA of organs from CINAP+/+ and CINAP+/− mice (female, 6 week of age, 3 mice used, randomly selected) was extracted and subjected to northern blot analysis with the 5′-ITS1 probe. 28S and 18S rRNA were shown as loading control. Killing of mice was approved by Peking University Laboratory Animal Center. (c) 35S-methionine labelling of CINAP+/+ and CINAP+/− MEF cells. The number of counts was normalized to protein content. *P<0.05 (Student’s t-test). (d) Cell proliferation rate of CINAP+/+ and CINAP+/− MEF cells was measured by MTT assays. (NS, no significance, two-way analysis of variance (ANOVA)). (e) The body weight of female and male mice (10 mice per group, randomly assigned) was recorded weekly from 30 to 60 days after birth. Results are presented as mean±s.d. (no significance, two-way ANOVA). (f) 35S-methonione labelling in MEF cells stimulated with 100 nM insulin. The methionine incorporation level of wild-type MEF cells without insulin stimulation was considered as 100%. *P<0.05 (Student’s t-test). (g) hCINAP knockdown efficiency was assessed by quantitative reverse transcriptase–PCR and western blotting. *P<0.05 and **P<0.01 (Student’s t-test). (h) Northern blot analysis with the 5′-ITS1 probe was performed to detect the accumulation of 18S rRNA precursors with hCINAP depletion. 28S and 18S rRNA were shown as the loading control. (i) 18S rRNA production was detected by northern blotting using 18S rRNA probe with hCINAP reduction. (j) Ribosome profiling was performed with MCF7 cells expressing control-shRNA or hCINAP-shRNA. Formation of 40S subunit was monitored by measuring the absorbance at 254 nm. Presence of hCINAP in each fraction was detected by western blot using anti-hCINAP antibody. (k) HeLa cells harbouring control-shRNA or hCINAP-shRNA were incubated with 1 μCi per ml 35S-Methionine and the incorporation of 35S-methonione was detected. **P<0.01 (Student’s t-test). (l) Cell proliferation assay was performed with HCT116 cells expressing control-shRNA or hCINAP-shRNA. Results (mean±s.d) were analysed by two-way ANOVA.
