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imb12072-sup-0001-si.eps8696K

Figure S1. Gene-silencing of Bombyx mori MHF provoked by dsRNA does not affect FancD2 monoubiquitination and cellular mytomycin C (MMC) sensitivity in BmN4-SID1 cells. (A) Semi-quantitative reverse transcription (RT)-PCR analysis of RNAi-mediated suppression of BmMHF1 and -2 mRNAs. Four relative positions of double-stranded (ds)RNA-targeted regions are indicated on a schematic representation of MHF1 and MHF2. The length of dsRNAs is shown in parentheses. The arrows indicate the positions of two sets of primers used for RT-PCR. PCR amplifications were performed on cDNAs obtained from BmN4-SID1 cells soaked in Dmc1, Rad51, Mhf1-1, Mhf1-2, Mhf2-1 or Mhf2-2 dsRNAs for 4 days. Transcript levels of the MHF genes were quantitated using ImageJ software. (B) Mhf depletion does not inhibit MMC-induced FancD2 monoubiquitination in BmN4-SID1 cells. BmN4-SID1 cells treated with dsRNAs for Mhf1 and -2 were treated with (+) or without (−) 40 μM MMC for 3 days, and visualized by immunoblotting for FLAG-tagged protein. The FancD2 mutation, FancD2K519R that is unable to monoubiquitinate was employed as a negative control. The FancD2 monoubiquitination was determined by mobility shift from the FancD2-S to the FancD2-L. The ratio of monoubiquitinated to nonubiquitinated FancD2 (L/S ratio) is shown below the lanes. (C) The depletion of Mhf does not sensitize silkworm cells to MMC. Cell proliferations of Mhf knockdown cells were assessed after treatment with MMC as described in (Fig. 3C). The data shown represent one of four independent experiments with similar result. Error bars represent the sd values of the means of triplicates.

imb12072-sup-0002-si.eps4382K

Figure S2. Gene-silencing of Bombyx mori RMI1 provoked by double-stranded (ds)RNA does not affect FancD2 monoubiquitination and cellular mytomycin C (MMC) sensitivity in BmN4-SID1 cells. (A) Semi-quantitative reverse transcription (RT)-PCR analysis of RNA interference-mediated suppression of BmRMI1 mRNA. Two relative positions of dsRNAs-targeted regions are indicated on a schematic representation of RMI1. The length of dsRNAs is shown in parentheses. The arrows indicate the positions of primers used for RT-PCR. PCR amplifications were performed on cDNAs obtained from BmN4-SID1 cells soaked in Dmc1, Rad51, Rmi1-1, or Rmi1-2 dsRNAs for 4 days. (B) Rmi1 depletion does not inhibit MMC-induced FancD2 monoubiquitination in BmN4-SID1 cells. BmN4-SID1 cells treated with dsRNAs for Rmi1 were treated with (+) or without (−) 40 μM MMC for 3 days, and visualized by immunoblotting for FLAG-tagged protein as described in (Fig. S1B). (C) The depletion of Rmi1 does not sensitize silkworm cells to MMC. Cell proliferations of Rmi1 knockdown cells were assessed following treatment with MMC as described in (Fig. S1C). The data shown represent one of four independent experiments with similar result. Error bars represent the sd values of the means of triplicates.

imb12072-sup-0003-si.eps4669K

Figure S3. Gene-silencing of Bombyx mori BLM provoked by dsRNA does not affect FancD2 monoubiquitination and cellular MMC sensitivity in BmN4-SID1 cells. (A) Semi-quantitative reverse transcription (RT)-PCR analysis of RNA interference-mediated suppression of putative BmBLM mRNA. Two relative positions of dsRNAs-targeted regions are indicated on a schematic representation of putative BLM. The length of dsRNAs is shown in parentheses. The arrows indicate the positions of primers used for RT-PCR. PCR amplifications were performed on cDNAs obtained from BmN4-SID1 cells soaked in Dmc1, Rad51, BLM-1 or BLM-2 dsRNAs for 4 days. (B) Blm depletion does not inhibit MMC-induced FancD2 monoubiquitination in BmN4-SID1 cells. BmN4-SID1 cells treated with dsRNAs for Blm were treated with (+) or without (−) 40 μM MMC for 3 days, and visualized by immunoblotting for FLAG-tagged protein as described in (Fig. S1B). (C) The depletion of Blm does not sensitize silkworm cells to MMC. Cell proliferations of Blm knockdown cells were assessed following treatment with MMC as described in (Fig. S1C). The data shown represent one of three independent experiments with similar result. Error bars represent the sd values of the means of triplicates.

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