Nachrichten aus der Brüder-Gemeine, 1828, no. 05

The Moravian Church's monthly journal of its worldwide activities, including in Labrador, published from 1819-94

Diagram of our hypothesis.

<p>hnRNP K normally binds to and stabilizes rRNA. A physiologic expression level of eIF3f competes with rRNA for the binding of hnRNP K. This contributes to the maintenance of the homeostasis of rRNA level and translation in cells. Increased eIF3f expression contributes to apoptosis via hnRNP K sequestration and increased rRNA degradation. On the other hand during tumorigenesis, decreased expression of eIF3f releases more hnRNP K to bind to rRNA, which leads to increased rRNA stability, translation and cell growth.</p

eIF3f induced rRNA degradation in pancreatic cells.

<p>(A) Restoration of eIF3f expression in pancreatic cancer cells decreased the rRNA level. MiaPaCa-2 cells were transfected with pcDNA3-eIF3f or pcDNA3. Relative fold changes of rRNA and eIF3f levels were quantified by real time RT-PCR and normalized to GAPDH mRNA. (B)(C) Silencing of eIF3f expression increased the rRNA level during apoptosis. Control and eIF3f-silenced HPDE cells were treated with DMSO vehicle control or etoposide (10 µM) for indicated time to induce apoptosis. Cells were harvested and total RNA was isolated using RNeasy Kit. Relative fold changes of 28S (B) and 18S (C) rRNA levels were quantified by real time RT-PCR after DNase treatment. (D) Time course of cell survival after etoposide treatment. HPDE cells were treated with DMSO or etoposide (10 µM) for indicated time. Cell survival was measured using MTT assay. Relative percentages of survived cells compared to DMSO-treated cells were shown. (E) Silencing of eIF3f expression attenuated rRNA degradation. Control and eIF3f-silenced HPDE cells were treated with actinomycin D (0.5 µg/ml) to block transcription for 3, 6 and 8 h before harvest. Total RNA was isolated, treated with DNase, and relative 28S and 18S rRNAs fold changes compared to control were quantified by real time RT-PCR and normalized to GAPDH mRNA.</p

eIF3f-silenced HPDE cells showed malignant features in 3D culture and soft agar assay.

<p>(A) Control and eIF3f-silenced HPDE cells were stably transduced with a GFP-expressing lentivirus (pLKO-puro CMV-TurboGFP, Sigma-Aldrich) and positive cells were sorted by a cell sorter (FACSAria) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034194#pone.0034194.s003" target="_blank">Fig. S3</a>). These GFP-expressing cells were used in an ex vivo 3D- cell culture system, immunofluorescent and confocal microscopy analysis. Laminin V (indicating basement membrane) was labeled with laminin V antibody and secondary antibody conjugated with Texas Red. A representative picture of our 3D cell culture system is shown. Note the loss of normal architecture, of cellular polarity, and of smooth basement membrane in eIF3f-silenced HPDE cells. Bar: 50 µm. (B) We counted regular sphere and irregular structures of control cell lines and 2 eIF3f-silenced HPDE cell lines after 10 days of 3D culture. Note that eIF3f-silenced cells formed much more irregular structures than control cells. (C) Soft agar assay: control and eIF3f-silenced HPDE cells were seeded at a density of 10000 cells per well in 6-well plate in 2 ml 0.33% agar and cultured for 14 days. Colonies were stained with 0.05% crystal violet overnight at 4°C. Colonies in the entire well were counted. Representative images of colonies and histogram is shown.</p

eIF3f inhibited both cap-dependent and IRES-dependent translation.

<p>(A) The map of the pRF bicistronic luciferase reporter (Renilla luciferase [Rluc], Firefly luciferase [Fluc]) constructs contained 1 of the 2 IRES elements of EMCV or HCV. (B) Same number of control and eIF3f-silenced HPDE cells were transfected with the same amount of 1 of the pRF bicistronic luciferase constructs. Dual luciferase assay for both firefly and renilla luciferase activity was performed 24 hours after transfection according to the manufacturer's instructions (Promega). Relative luciferase activity compared to control RNAi cells from 3 independent experiments was shown. (C) Control and eIF3f-silenced HPDE cells were either asynchronous or treated with nocodazole (40 ng/ml) for 16 h to block the cells in G2/M phase. Cells were harvested and Western blot was performed using CDK11, cyclin B1 and vinculin antibodies. Note that CDK11^p58 expression was present only in mitotically synchronized control cells, but was also present in asynchronous eIF3f-silenced cells because of increased IRES activity in its 5′UTR. Cyclin B1 expression confirmed the G2/M phase. Vinculin was used as loading control. (D) Starvation led to increased eIF3f protein level. HFF-1 and HPDE cells either were cultured in regular medium or were starved for 6 hours in PBS before harvest. Western blot is performed to determine eIF3f protein level. Densitometric analysis of the eIF3f bands normalized to corresponding α-tubulin is shown at the bottom.</p

eIF3f-silencing in normal pancreatic epithelial cells led to malignant transformation.

<p>(A) In HPDE cells, eIF3f-silenced cells had a higher proliferation rate. Same number of eIF3f stable knockdown (clone #5) and control HPDE cells (5×10⁴ cells/plate) were seeded in triplicate on 100-mm plates and total cell numbers were counted every 2–3 days. (B) eIF3f-silenced cells formed more colonies. Same number (1000 cells) of eIF3f stable knockdown and control HPDE cells were seeded on 100 mm plates and colony formation was measured after 14 days. (C–E) eIF3f-silenced cells migrated faster than control cells. Confluent eIF3f RNAi or control RNAi HPDE cells were used for a scratch assay and pictures were taken at the indicated time as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034194#s2" target="_blank">Materials and Methods</a> (C). Same cells were also used for a migration assay using a Transwell culture system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034194#s2" target="_blank">Materials and Methods</a>. A representative picture of the migrated cells stained with crystal violet on the filter was shown (D). Average number of migrated cells were counted and statistic difference between 2 cell lines was shown (E). (F) eIF3f-silenced cells were more resistant to apoptosis. eIF3f or control RNAi HPDE cells were treated with staurosporine (10 ng/ml) (<u>ST</u>) to trigger apoptosis. Apoptosis was measured after 24 h for ST using Annexin V staining and flow cytometry. Percentage of apoptotic cells out of total cells from 3 independent experiments was shown. (G) eIF3f-silenced cells were more resistant to gemcitabine treatment. eIF3f or control RNAi HPDE cells were treated with different concentrations of gemcitabine for 24 h before measuring the cell survival by MTT assay. The average ratio of gemcitabine/vehicle-treated cells OD from 3 independent experiments was plotted against gemcitabine concentrations.</p

Localization of eIF3f/hnRNP K and their relationships with P body and stress granule.

<p>HPDE or HFF-1 cells were treated with sodium arsenite (0.5 mM) for 45 minutes to trigger P bodies and stress granules formation. (A)(B) Under stress, eIF3f was predominantly co-localized with both P bodies and stress granules. Immunofluorescent study was performed using eIF3f, P body markers (Rck, Dcp1a), or stress granule marker eIF4G antibodies and FITC (green) or Texas Red (red) -tagged fluorescent secondary antibodies in HPDE cells. Arrowhead indicated co-localization foci (yellow). Arrow indicated non-P body, non-stress granule foci. (C) eIF3f was also localized to non-P body, non-stress granule perinuclear foci (arrow) in HPDE cells. Same experiments as (A) were performed. (D)(E) hnRNP K was localized in stress granules and unknown cytoplasmic foci (arrow), but not in P bodies. Immunofluorescent study was performed using hnRNP K (mouse), Rck (rabbit), or eIF4G (goat) antibodies and FITC-tagged anti-mouse (green), Texas Red-tagged anti-rabbit (red) or Cy3-tagged anti-goat secondary antibodies (red) in HFF-1 cells. Bar: 10 µm.</p

Increased endogenous interaction between eIF3f and hnRNP K under stress.

<p>(A) A375 cells were treated with staurosporine (ST) for the indicated time and cell fractionation was performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034194#pone.0034194-Shi3" target="_blank">[17]</a>. 500 µg of the lysates from the soluble (S) and pellet (P) fractions of the cells were immunoprecipitated with IgG or hnRNP K antibodies, followed by immunoblot with eIF3f and hnRNP K antibodies (top 2 panels). 50 µg of the same lysates were used for immunoblot with eIF3f, hnRNP K and α-tubulin antibodies without immunoprecipitation (bottom 3 panels). (B) HPDE cells were treated with DMSO or etoposide (Eto) (10 µM) for 24 hours. Cell lysates were immunoprecipitated with IgG or eIF3f antibodies, followed by immunoblot with eIF3f and hnRNP K antibodies (left 2 panels). The intensities of the bands were quantified using ImageJ software. The same cell lysates were used for immunoblot with eIF3f, hnRNP K and vinculin antibodies without immunoprecipitaiton (right 3 panels). (C) Immunofluorescent staining to eIF3f (FITC, green) and hnRNP K (Cy3, red) in HFF-1 fibroblasts treated with sodium arsenite (0.5 mM) for 30 minutes. (D) eIF3f directly interacted with hnRNP K. The left diagram shows full-length eIF3f and the location of the Mov34/JAB_MPN domain. Four GST-eIF3f truncation mutants were designed to localize the binding site of eIF3f with hnRNP K. GST pull-down assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034194#s2" target="_blank">Materials and Methods</a>.</p

COMP presence in fibrostenotic Crohn’s disease and unaffected intestine.

<p>H&E staining of fibrostenoic (panel a) demonstrating smooth muscle hypertrophy and tissue architectural changes typical of Crohn’s disease, compared to normal unaffected intestine (panel b). Immunohistochemical staining for COMP (red), with DAPI nuclear background staining (blue), reveals accumulation within the submucosa and distorted muscularis (panels c, e) compared to minimal COMP staining tissue in normal intestine (panels d,f).</p

Glycoproteins differentiating inflammatory and fibrostenotic phenotypes.

<p>Glycoproteins differentiating inflammatory and fibrostenotic phenotypes.</p