Panel A shows a bar graph depicting normalized RNA levels of Malat1 in different cell conditions. The x-axis represents different experimental conditions, including asynchronous, Nocodazole-treated, and Cycloheximide-treated cells, while the y-axis shows normalized RNA levels. Panel B contains images from smRNA-FISH showing Malat1 localization in KO+FL and KO+A cells, with arrows pointing to cells with cytoplasmic Malat1 signal. Panel C presents a violin plot of nuclear Malat1 mean intensity in cells with cytoplasmic Malat1, with the y-axis indicating the intensity in arbitrary units. Panel D is a schematic diagram outlining the experimental timeline for Doxycycline induction and mitotic synchronization. Panel E shows another bar graph of normalized Malat1 RNA levels at different time points post-release from mitotic arrest, with the x-axis indicating release times and the y-axis showing RNA levels. Panel F contains images from smRNA-FISH of Malat1 in KO plus FL and KO plus A cells at different time points post-release from mitotic arrest, with the fraction of cells with cytoplasmic Malat1 foci indicated. Panel G presents a violin plot of nuclear Malat1 mean intensity at different time points post-release, with the y-axis showing intensity in arbitrary units.
Redundant elements in Malat1 trigger postmitotic degradation. (A) qRT-PCR analysis of normalized Malat1 RNA levels in p53-deficient Malat1 WT and KO MEFs, rescued with Doxy-inducible FL (KO+FL) and ΔA (KO+ΔA) Malat1 constructs, cultured for 3 days in the presence of 1 μg/ml Doxy and harvested asynchronous or at indicated time points after release from mitotic arrest with 50 ng/ml Noc in the absence or presence of 50 μg/ml Chx, as described in Fig. 3 A. (B) smRNA-FISH of Malat1 (red) in asynchronous KO+FL and KO+ΔA cells, cultured in the presence of 1 μg/ml Doxy for 3 days. DAPI, DNA (blue). Arrows point to cells with cytoplasmic Malat1 signal. Scale bar: 20 μM. (C) Violin plot of nuclear Malat1 MI, measured in a.u., in KO+FL and KO+ΔA cells with cytoplasmic Malat1, quantified in images from B. (D) Schematic of the experiment, showing Doxy-dependent induction of FL and ΔA expression for 3 days, followed by removal of Doxy, mitotic synchronization, and release into G1 for indicated times. (E) qRT-PCR analysis of normalized Malat1 RNA levels in KO+FL and KO+ΔA, collected at indicted time points, as described in D. (F) smRNA-FISH of Malat1 (red) in KO+FL and KO+ΔA cells, fixed after 4 h of Noc treatment (0 h) or replated for indicated time following mitotic shake-off (2 and 4 h), as described in D. DNA, DAPI (blue). Scale bar: 20 μM. Note that, at 0 h, the mitotic cells were washed away during the fixation and staining procedure. Numbers indicate the fraction ± SD of cells with 50 or more cytoplasmic Malat1 foci from three biological replicates, with n > 100 cells scored per replicate. (G) Violin plot of nuclear Malat1 MI, measured in a.u., quantified in images from F. In this figure, bar graphs show mean ± SD of n = 3 technical replicates, validated in biological replicates (A and E). n indicates the number of nuclei represented in violin plots (C and G). Unpaired t test (A, C, E, and G), *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.