Lactobacillus Brevis SB8803 Induces Heat Shock Proteins, Activates P38 MAPK Pathway And

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Lactobacillus Brevis SB8803 Induces Heat Shock Proteins, Activates P38 MAPK Pathway And

Supplemental figure legends

Supplemental Figure 1. The expression of miR-18a in normal cells, colon cancer tissues and colon cancer cell lines

Real-time PCR showed that miR-18a is endogenously expressed in human colon cancer tissues (A) as well as all of the colon cancer cell lines examined, including Caco2/bbe,

HT29, HCT116, SKCO-1, SW480 and -620 cells (B) (n=4).

Supplemental Figure 2. Confirmation of the transfection efficacies

The transfection of double-stranded miR-18a using Sendai virus envelopes increased the expression of miR-18a in the colon cancer cell lines (n=3).

Supplemental Figure 3. The miR-18a* expression in SW620 cells transfected with double-stranded miR-18a

Real-time PCR showed that the miR-18a* expression did not change in SW620 cells transfected with double-stranded miR-18a at 48 hours after transfection (n=3).

Supplemental Figure 4. The effects of miR-18a on the proliferation of SW620 cells

The MTT assay revealed that miR-18a did not change the proliferation of the SW620 within 48 hr (n=5).

1 Supplemental Figure 5. The Ki-67 expression in the SW620 cells with and without the overexpression of miR-18a

Immunocytochemical staining showed the Ki-67 expression to not be significantly different between the cells with and without miR-18a overexpression (n=3).

Supplemental Figure 6. The telomere length of SW620 cells transfected with miR-

18a

The telomere length of SW620 cells was examined by a Southern blotting analysis (a telomere length assay). The assay revealed that the telomere length was shorter in the miR-18a-overexpressing cells than in the control cells (n=3).

Supplemental Figure 7. The expression of hnRNP A1 in colon cancer

Real-time PCR showed that the hnRNP A1 protein and mRNA were highly expressed in colon cancer tissues (A) and cell lines (B) (n=4).

Supplemental Figure 8. The overexpression of miR-18a does not change the translation of the mRNAs for cell growth- and apoptosis-associated molecules

The mRNAs of tumor growth- and apoptosis-associated molecules that were

2 estimated to be targets of miR-18a were selected from an analysis of microRNA sequences using a software program (Targetscan, http://www.targetscan.org/vert_50/). The mRNAs of seven molecules were identified as potential targets. A Western blotting analysis revealed that the expression levels of cyclin D2, RUNX1, NEDD9, ER-α, PTP4A3, IGF-1 and CD166 did not differ between miR-18a-overexpressing cells and control cells, suggesting that miR-18a does not influence the translation of the mRNAs for these cell growth- and apoptosis-related molecules (n=3).

Supplemental Figure 9. The expression levels of histone H1 and heat shock cognate 70 (Hsc 70) in the nuclear and cytosolic fractions

The Western blot analyses showed that histone H1 was detected equally in the nuclear fractions of scramble and miR-18a-transfected cells, while Hsc 70 was detected in the cytosolic fractions of both scramble and miR-18a-transfected cells.

Supplemental Figure 10. The results of the single isotope study

Following treatment with [3H]-glycine for 24 hours, an expression vector encoding hnRNP A1 and/or double-stranded miR-18a was transfected into SW620 cells. Next, the cell lysates were immunoprecipitated with anti-hnRNP A1 antibodies, and the

3 radioactivity of each group was measured. The 3H activity was significantly lower in the

SW620 cells transfected with miR-18a than in the control cells at 72 to 96 hours after transfection (n=3).

Supplemental Figure 11. The effects of a proteasome inhibitor on the degradation of HIF-1

A Western blotting analysis showed that HIF1, which is one of the major targets of proteasomal degradation, was increased following treatment with MG132. This indicates that MG132 inhibited the proteasomal degradation effectively.

Supplemental Figure 12. The efficacy of the knockdown of ATG7, p62 and BAG3

The transfection of siRNAs targeting ATG7, p62 and BAG3 decreased the expression of

ATG7, p62 and BAG3, respectively (n=3).

Supplemental Figure 13. miR-18a induces the autophagosomal degradation of hnRNP A1 in normal.

The siRNAs targeting ATG7 and p62 repressed the decrease in the hnRNP A1 expression induced by miR-18a (n=3).

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