Pol III is required for the production of milRs.

<p>A. DNA sequence of <i>milR-1</i> locus. B. Northern blot showing the level of small RNAs in indicated strains after indicated treatments. ML-60218 is Pol III specific inhibitor and DMSO is used as the control. The cultures were treated with ML-60218 before they were collected. The ethidium bromide-stained gel in the bottom panel shows equal loading of RNA samples. C. Northern blot result showing the levels of <i>pri-milR-1</i> in <i>dicer</i> double knockout strain in the presence of the inhibitor. D. qRT-PCR analyses showing that the production of pri-milRNAs for <i>milR-1</i>, <i>milR-2</i>, <i>milR-3</i> and <i>milR-4</i> was inhibited by ML-60218. rRNA level was used as the loading control in the qRT-PCR analysis. <i>β-tubulin</i> and a Pol III-transcribed tRNA was served as the negative and positive control, respectively. WT indicates the wild-type strain. The asterisks indicate <i>P</i> value<0.05. Error bars indicate S.D.</p

sj-pdf-1-imr-10.1177_03000605211062789 - Supplemental material for Effects of different sedatives/analgesics on stress responses in patients undergoing craniotomy and bone flap decompression

Supplemental material, sj-pdf-1-imr-10.1177_03000605211062789 for Effects of different sedatives/analgesics on stress responses in patients undergoing craniotomy and bone flap decompression by Qingduo Guo, Meina Ma, Qiuying Yang, Hong Yu, Xupeng Wang, Chunling Wu and Rui Li in Journal of International Medical Research</p

Pol III specifically binds to milR loci.

<p>ChIP assay results showing the binding of Pol III to the milR loci. A <i>tRNA</i> and the <i>β-tubulin</i> gene was used as the negative and positive control, respectively. The asterisk indicate <i>P</i> value<0.05. Error bars indicate S.D.</p

<i>milR-1</i> transcription is mediated by a non-conventional Pol III promoter.

<p>A. Northern blot analysis showing the levels of <i>milR-1</i> in the indicated strains. B. qRT-PCR analysis showing the reduction of <i>pri-milR-1</i> levels in the <i>mut1</i> and <i>mut2</i> strains. WT and <i>milR-1^ko</i> served as the positive and negative control, respectively. C. ChIP assays using the c-Myc antibody showing the reduced binding of Myc-RPC7 at the <i>milR-1</i> locus with the mutated TATA-like element. The asterisk indicates <i>P</i> value<0.05. Error bars indicate S.D.</p

Pol III knockdown results in the reduction of milRNA expression.

<p>A. Race tube assays showing the growth rates of the indicated strains in the presence or absence of QA. B. Northern blot analysis showing the levels of <i>rpc5</i>-specific siRNA in the indicated strains. C. qRT-PCR analysis showing the reduction of pri-milR levels when <i>rpc5</i> was silenced. rRNA level was used as the loading control for qRT-PCR. A Pol III-transcribed tRNA was served as a positive control. D. Northern blot showing the levels of <i>milR-1</i> and <i>milR-4</i> small RNAs.</p

The involvement of Pol II in milRNA production.

<p>A. Race tube assays showing the growth rates of the indicated strains in the presence or absence of QA. B. Northern blot analysis showing the levels of <i>rpb5</i>-specific siRNA in the indicated strains. C and D. qRT-PCR analysis results showing the reduction of <i>rpb5 and β-tubulin</i> level in ds<i>rpb5</i> strain in the presence of QA. The asterisks indicate <i>P</i> value<0.05. Error bars indicate S.D. E. Northern blot analysis showing the levels of <i>milR-1</i> and <i>milR-4</i> small RNAs. F. ChIP assay using c-Myc antibody showing the binding of Pol II to milR loci in the Myc-RPB6 strain. A <i>tRNA</i> and the <i>β-tubulin</i> gene was served as the negative control and positive control, respectively.</p

RNA sequencing of poly(A) RNA results showing the presence/absence of Pol II transcripts in the selected milR loci.

<p>Viewing window was set as 2000 nt. The vertical line in each panel indicates the location of the indicated <i>milR</i> gene.</p

RNase Z is required for <i>milR-4</i> processing.

<p>A. A Diagram showing the predicted secondary structure of <i>pri-milR-4</i>. B. RT-PCR analysis, which used a pair of primers indicated in A, showing the presence of transcript that spanning the two alanine tRNAs and <i>milR-4</i>. C. Race tube assays showing the growth rates of the indicated strains in the presence or absence of QA. D. Northern blot analysis showing the levels of <i>rnaseZ</i>-specific siRNA in the indicated strains. E. qRT-PCR analysis showing the reduction of <i>rnaseZ</i> mRNA level in the ds<i>rnaseZ</i> strain in the presence of QA. The asterisk indicates <i>P</i> value<0.05. Error bars indicate S.D. F. Northern blot analysis showing the levels of <i>milR-4</i> milRNAs in the indicated strains in the presence or absence of QA.</p

qPCR primer sequences.

<p>qPCR primer sequences.</p

DNA sequences of <i>milR-1-4</i>.

<p>The sequences of milRNA, milRNA* and the poly T sequences are indicated. TATA-like elements are underlined with single dashed lines. Putative A-boxes and B-boxes are underlined with double and triple dashed lines, respectively. The solid triangles in the <i>milR-1</i> sequence indicate the nucleotides mutated in our promoter analysis.</p