Journal: Disease Models & Mechanisms

Article Title: Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis

doi: 10.1242/dmm.049234

Figure Lengend Snippet: Inhibition of Nup98-96 leads to G1 bypass and cell cycle de-regulation. (A-D′) Using engrailed - Gal4 modified with a temperature-sensitive Gal80 ( en TS ), the indicated UAS-RNAis were expressed in the posterior wing disc from mid-L3 to 28 h after puparium formation (APF) at 28°C. The dotted lines indicate the pupal wing anterior–posterior (A-P) boundary. Panels are shown in single color in A′, B′, C′ and D′ and in similar panels in this and other figures. Nup98-96 inhibition increased the number of mitoses (indicated by phospho-Ser10 histone H3, PH3) and S phases [indicated by 5-ethynyl-2-deoxyuridine (EdU) labeling in the posterior wing], at stages when the wing is normally post-mitotic. (E,E′) Adult eyes from a heterozygous sensitized background expressing UAS-cyclin E ( CycE ) under the GMR - Gal4 promoter and GMR -driven P35 are shown. (F-H′) Adding in UAS-Nup98-96 RNAi enhanced eye size and folding (F,F′), and increased the number of cone cells and interommatidial cells, as shown by staining for the septate junction protein Discs large (Dlg; also known as Dlg1) (G-H′). (I-L′) Using en TS , the indicated UAS -RNAis were expressed in the posterior wing disc for 72 h prior to dissection of wandering L3 larvae. The dotted lines indicate the A-P boundary. Nup98-96 inhibition increased the number of mitoses and S phases in the posterior wing disc. The EdU experiment was performed multiple times with 5, 10 or 20 min of EdU labeling. Data and number of replicates from 5 min of EdU labeling are shown. Yellow arrowheads in K′ and L′ indicate the posterior zone of non-proliferating cells (ZNC), which is normally G1 arrested, but undergoes S phases when Nup98-96 is knocked down. (M) An EdU pulse for 1 h followed by a 7 h chase and PH3 staining was used to label mid-L3 wing disc cells that progress from S to M phase in ∼8 h. This experiment was repeated three times, with intervals of 6, 7 and 8 h chase. (N) Examples of PH3 (green)/EdU (magenta) double-labeled cells are shown (yellow arrowheads). (O) Quantification of double-labeled cells in the posterior:anterior compartments normalizes for EdU incorporation in each disc and provides an indication of cell cycling speed differences between compartments. RNAi to Nup98-96 increased cycling speed in the posterior wing disc (*** P <0.024; t -test with Welch's correction). Plots of individual biological replicates include mean±s.e.m. Yellow scale bars: 50 µm; white scale bar: 25 μm.

Article Snippet: The cells were incubated with the indicated siRNA for 72 h, then harvested for fixation and staining or lysed for western blotting. siRNAs used were as follows: Silencer Select Negative Control No. 1 (4390843, ThermoFisher Scientific); Nup98-96 siRNA#1, Silencer Pre-designed siRNA (AM16708, ThermoFisher Scientific); Nup98-96 siRNA#2, Silencer Select Pre-designed siRNA (4392420, ThermoFisher Scientific); Nup98-96 siRNA#3, Nup98 siRNA (sc-43436, Santa Cruz Biotechnology).

Techniques: Inhibition, Modification, Labeling, Expressing, Staining, Dissection

Journal: Disease Models & Mechanisms

Article Title: Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis

doi: 10.1242/dmm.049234

Figure Lengend Snippet: Inhibition of Nup98-96 leads to cell death and compensatory proliferation. (A-L′) Using en TS , the indicated UAS -RNAis were expressed in the posterior wing disc for 72 h prior to dissection of wandering L3 larvae (unless otherwise indicated). The dotted lines indicate the A-P boundary. (A-D′) Nup98-96 inhibition increased apoptosis in the posterior disc, as indicated by cleaved Death caspase-1 (DCP1). (E-F′) Co-expression of UAS-P35 with Nup98-96 RNAi led to tissue overgrowth (E,E′) and, by day 5, wing pouch duplication, outlined in yellow (F,F′). (G-H′) Nup-98-96 knockdown led to activation of JNK signaling as detected by phosphorylated JNK staining (pJNK). (I-N) Co-expression of a dominant-negative form of Drosophila JNK, Basket ( Bsk DN ) had variable effects on DCP1 staining and increased the ratio of PH3 labeling in posterior:anterior discs, although overall PH3 signal decreased with Bsk DN ( Fig. S2 ) (ns, not significant; * P <0.05, ** P <0.01, *** P <0.005; Welch's t -test comparisons). (O) Adult wings expressing the indicated transgenes with en TS . Co-expression of Bsk DN with Nup98-96 RNAi severely reduced the size of the posterior wing. Plots of individual biological replicates include mean±s.e.m. Scale bars: 100 µm.

Article Snippet: The cells were incubated with the indicated siRNA for 72 h, then harvested for fixation and staining or lysed for western blotting. siRNAs used were as follows: Silencer Select Negative Control No. 1 (4390843, ThermoFisher Scientific); Nup98-96 siRNA#1, Silencer Pre-designed siRNA (AM16708, ThermoFisher Scientific); Nup98-96 siRNA#2, Silencer Select Pre-designed siRNA (4392420, ThermoFisher Scientific); Nup98-96 siRNA#3, Nup98 siRNA (sc-43436, Santa Cruz Biotechnology).

Techniques: Inhibition, Dissection, Expressing, Activation Assay, Staining, Dominant Negative Mutation, Labeling

Journal: Disease Models & Mechanisms

Article Title: Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis

doi: 10.1242/dmm.049234

Figure Lengend Snippet: Inhibition of Nup98-96 leads to mispatterning, gene expression changes associated with wounding and a ‘loser’ phenotype. (A-H′) Using en TS , the indicated UAS -RNAis were expressed in the posterior wing disc for 72 h prior to dissection of wandering L3 larvae (unless otherwise indicated). Discs in C, D, G and H co-express P35 to block apoptosis and allow for tissue overgrowth. Samples in C and D were dissected after 5 days of Nup98-96 RNAi+ P35 expression. (A-D′) Wg levels are disrupted at the dorsal–ventral (D-V) margin but increased at the dorsal hinge upon Nup98-96 knockdown. The effect on Wg and wing disc overgrowth is enhanced by P35. (E-G′) Cut expression at the D-V margin is disrupted by Nup98-96 knockdown, independent of P35 expression. (H,H′) Vestigial (Vg) is reduced when Nup98-96 is knocked down. (I,J) RNAseq was performed on dissected late L3 wing discs expressing UAS-Nup98-96 or white RNAi for 72 h, driven by apterous- Gal4 with tub-Gal80 TS ( ap TS ). (I) A comparison of the overlap of genes significantly altered by Nup98-96 RNAi (0.5-log 2 fold or more) to previously published ‘wounding’ and ‘loser’ gene expression signatures in wings. The fold enrichment in the overlap of genes above that expected by chance is shown. (J) An M-A plot of the RNAseq data with significantly increased expression indicated in red and significantly decreased expression in blue. Genes in gray are not significantly altered. Scale bars: 100 µm.

Article Snippet: The cells were incubated with the indicated siRNA for 72 h, then harvested for fixation and staining or lysed for western blotting. siRNAs used were as follows: Silencer Select Negative Control No. 1 (4390843, ThermoFisher Scientific); Nup98-96 siRNA#1, Silencer Pre-designed siRNA (AM16708, ThermoFisher Scientific); Nup98-96 siRNA#2, Silencer Select Pre-designed siRNA (4392420, ThermoFisher Scientific); Nup98-96 siRNA#3, Nup98 siRNA (sc-43436, Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, Dissection, Blocking Assay

Journal: Disease Models & Mechanisms

Article Title: Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis

doi: 10.1242/dmm.049234

Figure Lengend Snippet: Knockdown of Nup98-96 leads to ribosomal protein mislocalization and compromised protein synthesis. (A-D′) Using en TS , the indicated UAS -RNAis were expressed in the posterior wing disc for 72 h prior to dissection of wandering L3 larvae and labeled for protein synthesis using O-propargyl-puromycin (puro) incorporation. Puro labeling experiments in discs were performed at multiple time points (10-20 min); data from one experiment with 12 min of labeling are shown. (E) The ratio of anterior:posterior puro-labeling is used to normalize for puro incorporation. Nup98-96 and Nmd3 knockdown reduced puro labeling (* P <0.05; unpaired Student's t -test). (F-G′) Knockdown of Nmd3 or CG4364 (Pescadillo homolog) for 48 h in the posterior wing disc using en TS activated JNK signaling. (H-K′) Using enRFP TS , the indicated UAS -RNAis were expressed for 72 h in backgrounds expressing GFP or YFP protein traps for the indicated Rp subunits. (K) RpL10Ab-YFP shows aberrant nuclear enrichment when Nup98-96 is knocked down. Plots of individual biological replicates include mean±s.e.m. Scale bars: 50 μm.

Article Snippet: The cells were incubated with the indicated siRNA for 72 h, then harvested for fixation and staining or lysed for western blotting. siRNAs used were as follows: Silencer Select Negative Control No. 1 (4390843, ThermoFisher Scientific); Nup98-96 siRNA#1, Silencer Pre-designed siRNA (AM16708, ThermoFisher Scientific); Nup98-96 siRNA#2, Silencer Select Pre-designed siRNA (4392420, ThermoFisher Scientific); Nup98-96 siRNA#3, Nup98 siRNA (sc-43436, Santa Cruz Biotechnology).

Techniques: Dissection, Labeling, Expressing

Journal: Disease Models & Mechanisms

Article Title: Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis

doi: 10.1242/dmm.049234

Figure Lengend Snippet: Knockdown of Nup98-96 in human cells leads to reduced protein synthesis and JNK signaling. (A-B′,D-E′,G-H′) PC3 cells were treated with small interfering (si)RNAs for 72 h, and cells were either fixed and stained with anti-Nup98 antibody (A-B′) or pJNK (D-E′), or labeled with puro for 12 min (G-H′). Control siRNA (ctrl) is a scrambled siRNA. (C,F,I) NUP98 siRNA reduces Nup98 levels (C) as well as reduces protein synthesis (F) and increases pJNK labeling (I). (J-L) Western blot analysis of PC3 cells treated with Ctrl and NUP98 siRNAs (L) shows that NUP98 siRNAs reduced the protein level of Nup98 (J) as well as increased phosphorylated JNK (K). Quantifications of fluorescence were performed on individual cells from three replicates from at least two independent experiments. Plots of individual biological replicates include mean±s.e.m. Quantifications for the western blots were done in triplicate for three different sets of siRNAs (* P <0.05, ** P <0.01, **** P <0.0001; unpaired Student's t -tests; F uses Welch's correction for unequal sample size). Scale bars: 10 μm.

Article Snippet: The cells were incubated with the indicated siRNA for 72 h, then harvested for fixation and staining or lysed for western blotting. siRNAs used were as follows: Silencer Select Negative Control No. 1 (4390843, ThermoFisher Scientific); Nup98-96 siRNA#1, Silencer Pre-designed siRNA (AM16708, ThermoFisher Scientific); Nup98-96 siRNA#2, Silencer Select Pre-designed siRNA (4392420, ThermoFisher Scientific); Nup98-96 siRNA#3, Nup98 siRNA (sc-43436, Santa Cruz Biotechnology).

Techniques: Staining, Labeling, Western Blot, Fluorescence

Journal: Disease Models & Mechanisms

Article Title: Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis

doi: 10.1242/dmm.049234

Figure Lengend Snippet: Overexpression of Nup98 disrupts protein synthesis and activates JNK signaling. (A-F) Using en TS , the indicated UAS -cDNA constructs were expressed in the posterior wing from mid-L2 and adult wings were mounted. Overexpression of Nup96 had no effect on the posterior wing, while overexpression of Nup98 or Nup98-96 reduced posterior wing size and disrupted vein patterning. Scale bars: 100 μm. (G-H′) Using en TS , a ubiquitous RFP-NLS was expressed with UAS-Nup98 2F for 24 h. The nuclear:cytoplasmic ratio for RFP-NLS was quantified and shown for the anterior wing disc (no Nup98 expression) and posterior wing disc (Nup98 overexpression). Ratios are also provided for Nup98-96 RNAi (from Fig. S4 ) for comparison. Scale bars: 10 μm. (I-J′) Using en TS , Nup98-96 cDNA was expressed in the posterior wing disc for 72 h prior to dissection of wandering L3 larvae and labeling with pJNK. UAS- white RNAi serves as a negative control, showing that endogenous pJNK at this stage is very low. Scale bars: 50 μm. (K-N′) Using en TS , the indicated UAS -cDNA or RNAi was expressed for 72 h prior to dissection and labeling with puro to measure protein synthesis. Overexpression of Nup98 2F reduced protein synthesis in the posterior disc, while Nup98-96 overexpression had a milder effect (ns, not significant; * P <0.05, *** P <0.005, **** P <0.0001; unpaired Student's t -test). Plots of individual biological replicates include mean±s.e.m. Scale bars: 50 μm

Article Snippet: The cells were incubated with the indicated siRNA for 72 h, then harvested for fixation and staining or lysed for western blotting. siRNAs used were as follows: Silencer Select Negative Control No. 1 (4390843, ThermoFisher Scientific); Nup98-96 siRNA#1, Silencer Pre-designed siRNA (AM16708, ThermoFisher Scientific); Nup98-96 siRNA#2, Silencer Select Pre-designed siRNA (4392420, ThermoFisher Scientific); Nup98-96 siRNA#3, Nup98 siRNA (sc-43436, Santa Cruz Biotechnology).

Techniques: Over Expression, Construct, Expressing, Dissection, Labeling, Negative Control