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A
(Left) Representative cross‐section images showing relative distribution between MATR3 and PcG proteins BMI‐1 and EZH2. (Right) Line charts showing pixel intensity of each channel on the ROIs. r represents coefficient of correlation. Scale bar, 5 μm.
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B
(Upper) Western blotting of anti‐MATR3 immunoprecipitation (anti‐IgG IP as negative control) products hybridized with MATR3, EZH2 and BMI‐1 antibodies. (Lower) Western blot of anti‐EZH2 immunoprecipitation (anti‐IgG IP as negative control) products hybridized with EZH2 and MATR3 antibodies.
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C, D
The representative cross‐section image showing nuclear distribution of BMI‐1 (C) and EZH2 (D) in Ctrl and shMatr3 (+Dox 3d) cells. Scale bar, 10 μm.
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E
(Upper) Standard deviation of BMI‐1 pixel intensity upon Ctrl (n = 86) and shMatr3 (+Dox 3d) (n = 89) cells. ****P < 0.0001, Student's t‐test. Error bars represent SD. (Lower) Standard deviation of EZH2 pixel intensity upon Ctrl (n = 86) and shMatr3 (+Dox 3d) (n = 97) cells. The P‐values were calculated using unpaired two‐tailed Student's t‐test; ****P < 0.0001. Error bars indicate mean ± s.e.m.
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F
Western blotting showing the distribution of BMI‐1 and EZH2 proteins in chromatin‐non‐associated and chromatin‐associated extracts before and after MATR3 knockdown (+Dox 3d) in AML12 cells. Representative of two independent replicates with similar results.
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G, H
(Left) The representative cross‐section image showing nuclear distribution of BMI‐1 (G) and EZH2 (H), and colocalization with H3K27me3 upon Ctrl and MATR3 knockdown (+Dox 3d). (Right) Line charts showing pixel intensity of each channel on the ROIs. r, coefficient of correlation Scale bar, 5 μm.
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I
The RNA level of Gapdh, Malat1, Neat1 and Hnf1aos1 in anti‐EZH2 RIP products relative to that in anti‐IgG RIP products as detected by RT–qPCR. For each sample, the relative RNA level was normalized to Actb. The RIP–qPCR assay was performed in six independent biological replicates (n = 6). The P‐values were calculated using unpaired two‐tailed Student's t‐test; ns, not significant. Error bars indicate mean ± s.d.
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J
(Upper) Western blotting showing the protein level of MATR3 and BMI‐1 in shCtrl and shBmi‐1 cells; (Lower) Western blotting showing the protein level of MATR3 and EZH2 in shCtrl and shEzh2 cells.
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K
The representative images showing nuclear colocalization of MATR3 with AS L1 RNA in shCtrl, shBMI‐1 and shEZH2 AML12 cells. Scales bar, 5 μm.
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L
Coefficient of correlation between MATR3 and AS L1 RNA in shCtrl (n = 42), shBMI‐1 (n = 48) and shEZH2 (n = 46) AML12 cells. The P‐values were calculated using unpaired two‐tailed Student's t‐test; ns, not significant. Error bars indicate mean ± s.e.m.
