EMBO/SourceData huggingface dataset:
Figure 3A Diagram of the dissected miniature pig mandible slice illustrating the deciduous (DC) and permanent (PC) canine and application of compression in vitro using Flexcell FX-5000 Compression System.B-D H&E staining of canine frontal sections from embryonic day 60 (E60) (B), after culturing for two days without stress (0 kPa) (C), or with stress (3 kPa) (D). (B'-D') are magnifications of boxed regions in their corresponding figure panels. Scale bars = 50 µm. n = 6/6 in E60 group; n = 12/12 in 0-kPa group; n = 4/8 in 3-kPa group.F RT-qPCR of several mechanosensitive proteins (TGFB1, NFKB1, EGR1, and RUNX2) and of four mechanoreceptors (ITGB1, ITGB3, ITGAV and ITGA2) in PC and surrounding soft tissue at E60 and after culturing for two days with (3 kPa) or without extra mechanical stress (0 kPa). n = 3 for each group. Data represent the means ± SEM. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups), *P < .05, **P < .01, ***P < .001; NS, not significant. =
Figure 4A-L IF of integrin β1, ERK1, and RUNX2 in miniature pig canine frontal sections at embryonic day 60 (E60), E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days; (A'-L') are magnifications of boxed regions in the corresponding figure panels. M Relative IF expression levels of integrin β1, ERK1, and RUNX2 during E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. Scale bars = 25 µm (A', G') and 50 µm (other panels). Unpaired t-tests for M , *P < .05, **P < .01, ***P < .001; NS, not significant.N Morphology of the human PC at weeks 18-19 (H&E staining); (N') is magnification of the boxed region in (N). DC, deciduous canine; PC, permanent canine; IEE, inner enamel epithelium; OEE, outer enamel epithelium. Mesenchyme between DC and PC is indicated with a green arrow.O-Q ISH expression patterns of ITGB1, ERK1, and RUNX2 in human PCs. Green arrows indicate the mesenchyme between DC and PC.R IF of RUNX2 in human PC. Mesenchyme between DC and PC is indicated with a red arrow. Red arrow indicates the mesenchyme between DC and PC.S PC epithelium areas in E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. , *P < .05, **P < .01, ***P < .001; NS, not significant.IF of RUNX2 and phospho-ERK1/2 (p-ERK1/2) upon force loading with 0 or 1.0 g/cm2 for 2 h or after force was removed and the cells cultured for an additional 1, 2, and 4 hours.V Relative IF expression levels of RUNX2 and p-ERK1/2 between groups Data information: Data represent the means ± SEM. n = 3. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for V , *P < .05, **P < .01, ***P < .001; NS, not significant.W Western blots of p-ERK1/2 and RUNX2 levels after 1.0 g/cm2 was applied for 2 h, with or without anti-integrin β1 antibody and ERK1 inhibitor (U0126). X Relative expression levels of p-ERK1/2, RUNX2, and Actin in (W). Data information: Data represent the means ± SEM. n = 3. Unpaired t-tests for M; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for X, *P < .05, **P < .01, ***P < .001; NS, not significant.
Figure 5A-C H&E staining of miniature pig canine frontal sections from embryonic day 60 (E60 group) (A) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag (overexpression group) (B) or overexpression control lentiviral vector with a Myc tag (O-control group) (C); (A'-C') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µmD-F Immunohistochemical (IHC) staining of Myc expression in mandible slices from the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 100 µm (D-F)G-I IF staining of RUNX2 expression (yellow arrows) in mandible slices of the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µmJ, K Relative expression levels of Myc and RUNX2 in the E60, O-control, and overexpression groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for J, K , *P < .05, **P < .01, ***P < .001.L, M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector (knockdown control, K-control) (L) or RUNX2 shRNA lentiviral vector (knockdown) (M); (L'-M') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm (other panels).N Relative RUNX2 expression levels between K-control and knockdown groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for N , *P < .05, **P < .01, ***P < .001.O-Q H&E staining of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (O'-Q') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µmR-T IF staining of RUNX2 expression (yellow arrows) of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (R'-T') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 25 µm (R'-T')U Relative RUNX2 expression levels among the three groups Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for U, *P < .05, **P < .01, ***P < .001.V PC epithelium areas in the overexpression, O-control, knockdown, knockdown + force, K-control, and K-control + force groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for V; , *P < .05, **P < .01, ***P < .001.
Figure 6A, B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 (E60) and E90. (A', B') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.C Relative ISH expression levels of RUNX2 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for C , *P < .05, **P < .01, ***P < .001.D, E ISH of miniature pig canine frontal sections showing expression patterns of β-catenin at E60 and E90. (D', E') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.F Relative ISH expression levels of β-catenin in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.G, H IF of Lef1 at E60 and E90. (G, H') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.I Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.J FISH of β-catenin in E60 and E90 mandible slices and E60 mandible slices after 12 and 24 h in culture via RNAscope. Boxed regions of mesenchyme are magnified in the right panels. Epithelium is indicated with a yellow arrow. Data information: Scale bars = 50 µm.K Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme at E60, E90, and E60 cultured for 12 and 24 h. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA for K, *P < .05, **P < .01, ***P < .001.L, M IF of β-catenin at E60 and E90; The yellow arrows indicate the localization of β-catenin in the nucleus. Data information: Scale bars = 50 µm.N Relative IF expression levels of β-catenin in the nucleus of epithelium and mesenchyme at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.O, P FISH of β-catenin in mandible slices infected with RUNX2 shRNA lentivirus (knockdown) or scrambled shRNA lentivirus (scramsh) via RNAscope. Data information: Scale bars = 50 µm.Q Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme in knockdown and scramsh groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.
Figure 7A, B IF of Lef1 in mandible slices at embryonic day 60 (E60) infected with RUNX2 overexpression lentiviral vector (overexpression) or overexpression control lentiviral vector (O-control). (A'-B') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. C Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) of the overexpression and O-control groups. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for C , **P < .01, ***P < .001; NS. not significant.D, E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days. (D', E') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. F Relative IF expression levels of Lef1 in the epithelium and mesenchyme after applying 3 and 0 kPa pressure. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for , *P < .05, **P < .01, ***P < .001; NS. not significant.G Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector. (G') Relative expression levels between control vector and RUNX2 overexpression groups. Data information: Data represent the means ± SEM. Unpaired t-tests , *P < .05, **P < .01, ***P < .001; NS. not significant.H Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with scrambled shRNA (scramsh) or RUNX2 knockdown shRNA (knockdown) lentiviral vectors. (H') Relative expression levels between scramsh and knockdown groups.I Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs of RUNX2 overexpression (O), control vector (V), RUNX2 knockdown (K), scramsh (S), compressed force (F; 1.0 g/cm2), and control (C; 0 g/cm2) groups. Sample were loaded in a gradient (7.5, 15, 30, and 60 μg) showing a linear relationship for each group.J Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs treated with IWR-1-endo. Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR-1-endo treatment.
Figure 1(C) Survival curves after the injection of EL4 lymphoma cells (n = 6/group).(H) Ifng protein and Ifng mRNA levels (n = 4/group) were determined by ELISA and quantitative PCR.
Figure 2(A) m6A dot blot assay using total RNA from NK92-siCTRL or NK92-siFTO cells.(D) Western blotting for the detection of GZMB in NK92-siCTRL and NK92-siFTO cells after treatment with IL-2.(E) Levels of IFNγ (n = 5 biological replicates) and TNFα (n = 3 biological replicates) in the culture media 24 hours after IL-2 treatment (left) or co-culture media with K562 cells for 4 hours (right) were determined by ELISA.
Figure 3(B) Western blotting analysis. NK92-siCTRL or NK92-siFTO cells were incubated with 20 ng/ml IL-2 for 24 hours, washed, and cultured without IL-2 (IL-2 depletion) for the indicated times (n = 3). (C) A western blotting analysis of NK92+MOCK or NK92+FTO cells incubated with 20 ng/ml IL-2 for the indicated times was performed (n = 3). D
Figure 4(C) m6A methylated RNA immunoprecipitation & quantitative PCR (MeRIP-qPCR) analysis using a m6A antibody in NK92-siFTO (upper) or NK92+FTO (lower) cells compared to NK92-siCTRL or NK92+MOCK cells.(D) SOCS3 3'-UTR reporter assay using the AANAT reporter system. Plasmid information for the AANAT reporter system with or without the SOCS3 3'-UTR sequence (upper). Levels of Aanat protein (left) and Aanat mRNA (right) expression in HEK-293T cells co-transfected with plasmid-expressing AANAT reporter containing SOCS3 3'-UTR and siCTRL or siFTO.
Figure 5(B) Relative mRNA expression levels of JAK/STAT target genes, IFNγ (n = 10 biological replicates), GZMB (n = 6 biological replicates) and PRF1 (n = 8 biological replicates) were determined by qPCR in CD56+-siCTRL or CD56+-siFTO cells.(C) Western blotting of CD56+-siCTRL or CD56+-siFTO cells incubated with 20 ng/ml IL-15 and 20 ng/ml IL-21 for the indicated times was performed. Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated (p-) and total proteins (n = 2~3). Data are representative of three times independent experiments with similar results. The protein levels were expressed as the relative band density of the corresponding protein (lower).
Figure 6(E) The images of the ventral bioluminescence (BLI) following the injection of NK92-TRC or NK92-shFTO cells.(G) The survival curves after the injection of K562-Luc cells.
Figure 1Negative stain TEM image of B. cereus NVH 0075-95 endospore, showing spore body (SB), exosporium (E), and endospore appendages (Ena), which emerge from the endospore individually or as fiber bundles. At the distal end, Enas terminate in a single or multiple thin ruffles (R).cryoEM images (left) and negative stain 2D class averages of single S-Ena (E) and L-Ena (F) fibers. S-Ena show ~100 Å diameter and a helical or staggered appearance with a ~37 Å rise, whereas L-Ena have a ~80 Å diameter and appear as stacked disks of ~40 Å height and ~60 Å translation along the fiber. L- and S-Ena show, respectively, a single or multiple ruffle(s) (black arrow, labeled 'R') at the distal end of fiber.
Figure 3SDS-PAGE of recEna1B, treated with β-mercaptoethanol or TEV protease (to remove N-terminal 6xHis tag) as indicated. Bands with apparent MW of ~13 and ~15 kDa correspond recEna1B monomer with (Ena1B6His) and without (Ena1BC) the 6xHis tag and TEV recognition site, respectively. In the uncleaved sample, a band running at ~ 30 kDa (labeled *) corresponds to a non-physiological disulfide bound S-Ena1B dimer. Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS-resistant high molecular weight complexes stuck in the stacking gel.Negative stain TEM images of rec1Ena1B oligomers formed after refolding, but prior to TEV removal of the N-terminal 6xHis tag. Close-up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S-Ena fiber (model - right). Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs.
Figure 5Transcription level of ena1A (x), ena1B (▲), ena1C (○) and dedA (♦) relative to rpoB determined by RT-qPCR during 16 hours of growth of B. cereus strain NVH 0075-95. The dotted line represents the bacterial growth measured by increase in OD600. Of note, the transcription of ena1C was surprisingly higher than ena1A and ena1B, the major components of the isolated S-Ena (Fig EV2).
Figure 6Representative negative stain images of endospores of NVH 0075-95 mutants lacking ena1A, ena1B, or ena1C, as well as endospores of strains complemented with the respective ena subunit expressed from plasmid (i.e. pA, pAB and pC). The ena1B mutant was complemented with a plasmid carrying both ena1A and 1B (pAB) due to repeated failure to transform with a plasmid holding ena1B only. Inset are 2D class averages of Enas observed on the respective mutants. Knockout of ena1A, ena1B, or ena1C results in the loss of S-Ena, a phenotype that is restored by plasmid-based complementation.Number (top) and length (bottom) of Enas found on WT, mutant (∆ena1A, ∆ena1B, or ∆ena1C) and plasmid complemented (∆ena1A:pena1A, ∆ena1B:pena1AB, or ∆ena1C:pena1C) NVH 0075-95 endospores. Statistics: pair-wise Mann-Whitney U tests against WT (n: ≥18 spores; n: ≥50 Enas; ns: not significant, * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001. ---: mean ± s.d.)
Figure 1A, B MAGE-A3/6 protein levels decrease upon EBSS treatment of HeLa cells for the indicated times. Note both bands are MAGE-A3/6. Quantitation represents average of n=3 with standard deviation shown.C Quantitative TMT proteomics of A375 cells grown in complete media (CM) or EBSS for 3 hrs. Number of proteins affected are shown. Autophagy related proteins are indicated in grey and MAGE-A proteins in blue.D, E MAGE-A3/6 protein levels are actively degraded upon nutrient deprivation. HeLa cells were treated for the indicated times with complete media (DMEM + 10% FBS) or EBSS containing 100 µg/mL translation inhibitor cycloheximide (CHX). Protein degradation rates are shown (E). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01. ns indicates p>0.05 using 2-way ANOVA.
Figure 2FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown.FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.C Removal of FBS from normal growth conditions (DMEM+10% FBS) results in MAGE-A3/6 protein degradation in HeLa cells.D, E MAGE-A3/6 protein stability is controlled by a <3 kDa, heat stable, lipophilic molecule present in FBS. HeLa cells were treated with EBSS alone, or EBSS containing 10% complete FBS, 10% charcoal-dextran stripped FBS, 10% boiled FBS, 10% dialyzed >3 kDa FBS, or 10% dialyzed <3 kDa FBS for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.F MAGE-A3/6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0.2 ng/mL, testosterone, or bovine pituitary extract 25 µg/mL.Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA (10 mM) or insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown., H Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.
Figure 3A MAGE-A3/6 mRNA levels do not change upon nutrient deprivation. RNA was isolated from HeLa cells treated with EBSS for the indicated times. RT-QPCR analysis was performed to detect both MAGE-A3/6 with a common primer set. Data was normalized to 18S rRNA levels. Quantitation represents average of n=3 with standard deviation shown.B, Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM).C Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.D Nutrient deprivation increases ubiquitination of endogenous MAGE-A3/6. HeLa cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.E MAGE-A3/6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase, TRIM28. HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times.F, MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times.G MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.H Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex. HeLa cells were transfected with vector control or the indicated dominant negative (D/N) Cullin constructs for 48 hrs before harvesting cells after 0 or 4 hrs EBSS treatment.I Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on Cul4A/B. HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B. 72 hrs after treatment, cells were incubated for the indicated times with EBSS before harvesting and immunoblotting. Note, time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.