Panel A: A line graph showing the population doublings of two wild-type mouse embryonic fibroblast (MEF) lines, WT1 and WT2, over 10 passages. The x-axis represents passage time in days, and the y-axis represents population doublings. Panel B: A bar graph showing qRT-PCR analysis of normalized Cdkn1a/p21 RNA levels in cells at passages 4 and 10 for WT1 and WT2. The x-axis represents the passage number, and the y-axis represents RNA levels. Panel C: A bar graph showing qRT-PCR analysis of normalized Malat1 RNA levels in cells at passages 2 and 10 for WT1 and WT2. The x-axis represents the passage number, and the y-axis represents RNA levels. Panel D: A standard curve for copy number calculation using dsDNA templates, generated from genomic DNA with Malat1 primer sets A, B, and E. The x-axis represents the copy number, and the y-axis represents Ct (Y). A table below shows the number of copies per cell in WT1 MEFs at passage 2, detected with indicated primer sets. Panel E: Microscopic images showing smRNA-FISH/IF detection of Malat1 (red) and nuclear speckle marker, Srrm2 (green), in WT1 and KO MEFs at passages 4 and 10. DAPI stains DNA (blue). Scale bar is 10 micrometers. Panel F: Microscopic image showing smRNA-FISH detection of MALAT1 (red) in RPE cells. DAPI stains DNA (blue). Scale bar is 20 micrometers. Panel G: A violin plot showing the quantification of nuclear MALAT1 mean intensity (MI) in cells with (early G1) and without (G1/S/G2) cytoplasmic Malat1 in images from Panel F. The x-axis represents cell cycle stages, and the y-axis represents nuclear MALAT1 MI in arbitrary units. Panel H: Microscopic image showing smRNA-FISH detection of MALAT1 (red) in PC9 cells. DAPI stains DNA (blue). Scale bar is 20 micrometers. Panel I: Microscopic images showing smRNA-FISH visualization of MALAT1 (red) across different cell cycle stages in RPE cells. DAPI stains DNA (blue). Scale bar is 10 micrometers. A schematic below shows representative MALAT1/DAPI patterns in mitosis (M), early G1, and G1/S/G2 cell cycle stages. Panel J: Microscopic images showing smRNA-FISH visualization of MALAT1 (red) across different cell cycle stages in PC9 cells. DAPI stains DNA (blue). Scale bar is 10 micrometers. A schematic below shows representative MALAT1/DAPI patterns in mitosis (M), early G1, and G1/S/G2 cell cycle stages.
Malat1/MALAT1 abundance and localization in murine and human cells, related to Figs. 1 and 2. (A) Growth curve of two independent primary WT MEF lines, WT1 and WT2, over 10 passages. (B) qRT-PCR analysis of normalized Cdkn1a/p21 RNA levels in cells from A at indicated passages. (C) qRT-PCR analysis of normalized Malat1 RNA levels in cells from A at indicated passages, using a primer set specific to region D, excludes production of truncated Malat1. (D) Top, standard curve for copy number calculation using dsDNA templates, generated from genomic DNA with Malat1 primer sets A, B, and E (Fig. 1 A); Bottom, number of copies per cell in WT1 MEFs at passage 2, detected with the indicated primer set. Unprocessed Malat1, detected with primer set E, represents < 1/200th (0.5%) of total Malat1, detected with primer sets A and B. (E) smRNA-FISH/IF detection of Malat1 (red) and nuclear speckle marker, Srrm2 (green), in WT1 and KO MEFs at indicated passages. DAPI, DNA (blue). Scale bar: 10 μM. Representative images illustrate differences in Malat1 abundance between passages, independent of speckles. (F) smRNA-FISH detection of MALAT1 (red) in RPE cells. DAPI, DNA (blue). Scale bar: 20 μM. * indicate cells in early G1 with cytoplasmic MALAT1 signal. (G) Quantification of nuclear MALAT1 mean intensity (MI), measured in a.u., in cells with (early G1) and without (G1/S/G2) cytoplasmic Malat1 in images from (F). (H) smRNA-FISH detection of MALAT1 (red) in PC9 cells. DAPI, DNA (blue). Scale bar: 20 μM. * indicate cells in early G1 with cytoplasmic MALAT1 signal. (I and J) Top, smRNA-FISH visualization of MALAT1 (red) across different cell cycle stages in RPE (I) and PC9 (J) cells. DAPI, DNA (blue). Scale bar: 10 μM. Bottom, schematic of representative MALAT1/DAPI patterns in mitosis (M), early G1, and G1/S/G2 cell cycle stages. In this figure, bar graphs show individual values and mean ± SD of n = 3 technical replicates (B and C). n indicates the number of quantified nuclei represented in violin plots (G). Unpaired t test (B, C, and G), **P < 0.01; ***P < 0.001.