EMBO/SourceData huggingface dataset:
Figure 6(A) UMUC3-PLKO and UMUC3-shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model, and cisplatin (1 mg/kg) was intraperitoneally administered. Images of tumors are shown after mice were sacrificed (N= 6). (B) Compared with the vector (PLKO) group, the tumor growth rate was significantly inhibited in shcircFNTA nude mice. (C) Tumor weights also decreased in the shcircFNTA group. (D-E) The qRT-PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced (D), and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group (E).(F) Representative immunohistochemistry (IHC) images detecting FNTA, p-ERK1/2 and p-MEK1/2 in PLKO tumurs as compared to shcircFNTA xenografted tumors. Scale bars: 100 μm (upper panels) and 25 μm (lower panels). (G) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA, p-ERK1/2 and p-MEK1/2. (H) The tumor tail vein injection model was established to observe lung metastasis (N=6). Representative images of lung metastasis foci in gross and microscopic HE examination. Scale bars: 400 μm (upper panels) and 100 μm (lower panels). (I) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group. (J) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group.
Figure 1(A) Autophagic markers from mixed midbrain gba+/+, gba+/−, and gba−/− cultures were analyzed via western blotting using antibodies as indicated.(B) Autophagic flux was analyzed in gba+/+ and gba−/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control.(C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. *p < 0.05.(D) Accumulation of p62/SQSTM1 was analyzed via immunoblotting. β-actin was used as a loading control.(E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies.(F) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated. β-actin was used as a loading control.(G) Proteasomal activity (chymotrypsin activity) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase. RFU, relative luciferase units. Data represent the mean ± SEM (n = 3, each experiment contained triplicates for each genotype). *p < 0.05.
Figure 2(A) Levels of soluble and insoluble α-synuclein from midbrain of P1 gba+/+ and gba−/− mice. TX-100 soluble and insoluble (SDS/Urea) factions were isolated and analyzed via western blotting with α-synuclein antibodies. β-actin was used as a loading control.(B) Sagittal brain stem sections from gba+/+, gba+/−, and gba−/− mice were stained with α-synuclein antibodies. Scale bar, 1,000 μm.
Figure 3(A) Neurons and astrocytes from gba+/+, gba+/−, and gba−/− mice were stained with TMRM. The mean florescence intensity in mitochondria was analyzed via confocal microscopy (n = 3, >32 cells analyzed/experiment).(B) Neurons from gba+/+ and gba−/− bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 1 min, 1 μM oligomycin was added followed by 1 μM FCCP (n = 5, three cells analyzed/experiment).(C) Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).(D) As in (C), except cells were bathed in 10 mM methyl succinate, (n = 3, four cells/experiment). All data in this figure represent the mean ± SEM, *p < 0.05. Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).
Figure 4(A) Oxygen consumption rates of gba+/+, gba+/−, and gba−/− mixed midbrain cultures. Basal oxygen consumption was measured over 3 min. The maximal (uncoupled) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A (n = 3, three runs/experiment).(B) Complex I-NADH: Ubiquinone reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).(C) Complex II-III: Succinate dehydeogenase, cytochome c reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).(D) Complex IV activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).(E) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy (n = 3, >16 cells/experiment).(F) gba+/+and gba−/− cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies. All data in this figure represent the mean ± SEM, *p < 0.05.
Figure 5(A) Neurons and astrocytes were immunostained for cytochrome c and stained with Hoechst. Far left panels were additionally immunostained for GFAP and MAP2 to mark neurons and astrocytes. "A" in nucleus denotes astrocytes. Scale bar, 20 μm.(B) Mitochondrial morphology was blind counted from cells in (A) with neurons scored as MAP2 positive and astrocytes as GFAP positive. A "normal" morphology is classified as a mixture of fused and fragmented mitochondria that comprise a wild-type network. (n = 3; 100 cells counted/experiment).(C) Levels of morphology proteins from gba+/+, gba+/−, and gba−/− isolated mitochondria were analyzed via immunoblotting with the indicated antibodies.(D) Midbrain gba+/+, gba+/−, and gba−/− astrocytes expressing GFP-DRP1K38A were immunostained for cytochrome c and mitochondrial morphologies analyzed; concurrently cells were stained with TMRM and fluorescence intensity analyzed via confocal microscopy. Scale bar, 20 μm (n = 3, >15 cells/experiment). All data in this figure represent the mean ± SEM.
Figure 6(A) Midbrain astrocytes were transfected with GFP-LC3, treated with 100 nM bafilomycin A1, and stained with MitoTracker Red and imaged using confocal microscopy.(B) Midbrain gba+/+ and gba−/− neurons and astrocytes were transfected with YFP-Parkin. Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy. Scale bar, 20 μm.(C) TMRM fluorescence intensity of cells from (B) was analyzed via confocal microscopy. Data represent the mean ± SEM, (n = 3, >4 cells analyzed per experiment).(D) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr. PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba+/+ and gba−/− neurons. Mitochondrial CI subunit NDUFS4 was used as a loading control.
Figure 4(A) Representative images of immunofluorescence analysis (with anti-PlxnB2 antibodies) of the phenotype of COS7 cells transfected with wild-type or mutated PlxnB2, compared to the collapsed phenotype induced by the cognate ligand Sema4C (1 μg/ml) in cells expressing the WT receptor. (B) The stacked bar graph shows the fraction of collapsed PlxnB2-expressing cells, identified as having a surface minor of 2000 μm2 (measured by ImageJ software), out of 50-100 cells counted per each condition, from different fields and n>3 experiments). The statistical analysis was performed comparing WT untreated cells with all other groups, by Fisher's Exact Test, which indicated a *****p value < 0.00001 for Sema4C-treated and for G842C-PlxnB2-expressing cells (confirmed by ANOVA analysis on individual cell values, shown in Appendix Fig. S2). By Fisher's test, the only other mutants significantly different from WT were R820H (p< 0.001) and R843Q (p<0.01), although ANOVA analysis on individual cell values shown in Appendix Fig. S2 didn't confirm such significance for other PlxnB2 variants.
Figure 5(A) The graphs show the analysis of cellular viability in shPlxnB2-tranduced agnospheres, compared to their respective scramble-transfected controls, after 24, 48, 72, and 96 hours of growth in culture. Values are mean ± SD of n≥3 independent experiments (6 technical replicates for each). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons (Graph Pad Prism 8.0.0): **p = 0.0053; ****p < 0.0001.(C) After transplantation in immunodeficient mice of CUP cells described above, the volume of ADX43- and (D) ADX901-shPlxnB2 tumors was assessed, in comparison with the respective controls. Plotted values indicate the mean ± SD (n = 8 each group). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons: **p = 0.0060; ****p < 0.0001.
Figure 6(A) Time course analysis of the cellular viability of AS901, stably overexpressing either wild-type or G842C-mutated PlxnB2, or controls transduced with an empty vector. Plotted values are the mean ± SD of n =3 independent experiments (six technical replicates for each). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs. controls (CTRL): at 24h **p = 0.0097, 48h **p = 0.0026, 72h **p = 0.0079, 96h *p = 0.0226. PlxnB2-G842C vs. PlxnB2-WT: at 48h $p = 0.0418, 72h $p = 0.0318. (B) Tumor growth curves of ADX 901 over-expressing either PlxnB2-G842C or PlxnB2-WT, or mock control. Plotted values are the mean ± SD (n = 8 each groups). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs control (CTRL): at 25 days **p = 0.0029, 28 days **p = 0.0028, 32 days **p = 0.0033. PlxnB2-G842C vs PlxnB2-WT: at 25 days $p = 0.0221, 28 days $p = 0.0234, 32 days $$p = 0.0024.
Figure 7(A) Western blotting showing basal endogenous or overexpressed PlxnB2, either WT or G842C-mutated isoforms, in MCF7 cells. In this wide view of the hybridized membrane, it is appreciable the presence of full length molecules at 250 kDa, as well as the processed 80 kDa fragments (reported in ref. 64). Vinculin staining provided protein loading controls.(B) The graph shows the mean ± SD of relative increase in 24-hour spontaneous migration across Transwell inserts of MCF7 cells (analysed above) over-expressing either wild-type or G842C-mutated PlxnB2, versus mock-transfected controls (n=4 biological replicates). The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: PlxnB2-WT vs. mock *p=0.0145; PlxnB2-G842C vs. PlxnB2-WT *p=0.0277; PlxnB2-G842C vs. mock ***p=0.0002.(C) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above. The graph shows the mean ± SD of values obtained in N=3 independent experiments (with at least three technical replicates for each). The statistical significance was verified by one-way ANOVA with Tukey's correction for multiple comparisons: *p = 0.0350; **p = 0.0029; ***p = 0.0002. Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B.(D) Western blotting analysis of endogenous and overexpressed PlxnB2 (either WT or G842C-mutated isoforms) in CUP cells AS906 (both full length molecules at 250 kDa and processed 80 kDa fragments). Vinculin staining provided protein loading controls.(E) The invasive capacity of CUP cells AS906 (shown in previous panel), overexpressing WT or G842-mutated PlxnB2 (or mock control), was assessed across Matrigel-coated transwell inserts. The graph shows the mean ± SD of values obtained in N=3 independent experiments (including technical duplicates, in two cases). Below the graph, are shown representative fields of the semipermeable membranes (additional larger size images can be found in appendix Figure S4C); scale bar: 100μm. The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: *p=0.0406; **p=0.0045; ***p=0.0003.
Figure 8(A) Phospho-EGFR analysis by western blotting in control and PlxnB2-silenced AS43 cells. Images show representative results of n=3 independent experiments, while the graph at the bottom shows the mean ± SD of normalized band quantification. The statistical significance across replicates was verified by unpaired t-test: ****p<0.0001. (B) Phospho-EGFR analysis by western blotting in AS901 and AS906 cells either mock, or overexpressing WT or G842C-mutated PlxnB2. Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by one-way ANOVA followed by Tukey test: AS901 PlxnB2-G842C vs. PlxnB2-WT *p=0.0434, PlxnB2-G842C vs. mock *p=0.0211; AS906 PlxnB2-G842C vs. PlxnB2-WT *p=0.0436, PlxnB2-G842C vs. mock *p=0.0481.(C) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres, after 6-hour treatment with the EGFR inhibitor cetuximab (50μg/ml). Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by Sidak's multiple comparison test between cetuximab-treated and untreated conditions, per each group: AS43 *p=0.0153, ***p=0.0001, ****p<0.0001; AS901 *p=0.0201.
Figure 9(A) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab (50μg/ml). In the plot, each box represents values from 25th to 75th percentiles, the central band indicates the mean, and the whiskers expand from maximum to minimum values of individual cetuximab-treated experimental points (n≥9), normalized to the average value of untreated cells within each group. The statistical significance across three independent experiments, each comprising ≥3 technical replicates, was verified by unpaired t-test multiple comparisons of cetuximab-treated and untreated conditions, per each group: ***p=0.0002; ****p<0.0001.(B) The viability of AS43, in presence (or absence) of cetuximab (50μg/ml), was assessed over the indicated time course, compared to values at T0. The graph shows the mean ± SD of two independent experiments (with quadruplicate technical replicates each). The statistical significance was verified through multiple comparisons by two-way ANOVA with Bonferroni correction of cetuximab-treated and untreated samples, per each time point: ****p<0.0001.(D) The invasive capacity of AS906 cells (overexpressing WT or G842C-mutated PlxnB2, or mock transfected) was assessed as in Fig. 7E, in the presence or absence of cetuximab 50μg/ml. Images show representative results (scale bar: 100μm), while the graph on the right shows the mean ± SD of n=3 independent experiments. The statistical significance across replicates was verified by one-way ANOVA with Bonferroni correction: **p= 0.0010; ***p=0.0005; ****p<0.0001.
Figure 1(A) Tandem affinity purification was used to isolate KDF1 associated proteins from SILAC-labelled keratinocytes. Precipitated proteins were resolved by SDS-PAGE and subjected to identification with LC-MS/MS. Arrows denote IgG heavy and light chains. Star denotes KDF1. Ctrl: control. Kd: kilodalton for molecular weight markers.(B-C) HEK293 cells were transfected with empty vector or plasmids encoding HA tagged KDF1 and/or GST-tagged IKKα. Immunoprecipitation (α-HA or α-GST) was carried out to determine their interaction. Immunoprecipitates (IP) and whole cell lysate (WCL) were immunoblotted (IB) with different antibodies as indicated.(D) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca). IPs were blotted with different antibodies as indicated. Star denotes KDF1.(E) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its kinase dead (KD) mutant. IP and WCL were blotted with different antibodies as indicated.(F) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its different truncation mutants (EV Fig. 1B). IP and WCL were blotted with different antibodies as indicated. Arrow denotes antibody heavy chain.(G) HEK293 cells were transfected to co-express GST-tagged IKKα with HA-tagged KDF1 or its different truncation mutants (EV Fig. 1C). IP and WCL were blotted with different antibodies as indicated.
Figure 3A) Immunoblots of WCL from IKKα KO cells with rescued expression of HA-tagged IKKα or its mutant using different antibodies as indicated.B) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated. Expression f exogenous KDF1 was induced by doxycycline (DOX) at different concentration.C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05(C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05E) Sections of regenerated skin developed from engrafted IKKα KO, KDF1 KO, and their rescued cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm.
Figure 4(A) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated. Lo: low calcium; Hi: high calcium.(B-C) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide (CHX). WCL was collected at 0, 30, 60, 90, 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody (B). Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified (C). Statistical analysis is conducted using 2-way ANOVA. N=3 (biological replicates). *, p<0.05. Error bar represents S.D. (standard deviation).(D-E) WT and KDF1 KO keratinocyte (right panels) or cells transfected with plasmid encoding exogenous IKKα (left panels) were treated with MG132 at 10µM for 6 hours, then were subjected to immunoprecipitation using anti-ubiquitin (Ub) antibody. IP and WCL were analyzed by immunoblots with α-IKKα antibody (D). Overall intensity of all ubiquitinated IKKα bands was determined by densitometry. Ratio of ubiquitinated IKKα was quantified and presented as bar graphs (E). Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). *, p<0.05. Kd: kilodalton for molecular weight markers.(F) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant, under both low and high calcium conditions, was examined and quantified by western blotting(G) PiggyBac transposon was used to ectopically express HA-tagged IKKα in KDF1 deficient cells. WCL were collected and analyzed by immunoblots with different antibodies as indicated.(H) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification. Skin sections from grafted tissue were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bars = 50 µm.
Figure 5A) HEK293 cells were transfected to co-express KDF1 with USP7. IP and WCL were blotted with different antibodies as indicated.B) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca), same as in Fig. 1D. IPs were blotted with different antibodies as indicated. The star denotes KDF1.C) WCL from WT and USP7 CRISPR KO cells were subjected to immunoblotting with different antibodies as indicated.D) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift.E) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours, then subjected to immunoprecipitation using anti-ubiquitin antibody. Precipitated product was analyzed by immunoblotting with IKKα antibody. Band intensity was determined by densitometry and shown as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). **, p<0.01. Kd: kilodalton for molecular weight markers.F-G) Immunoblot of Krt10 and Loricrin (Lor) with WT and USP7 KO cells before and after calcium shift (F). Star denotes an unspecific band in α-Loricrin blots. Band intensity was determined by densitometry and shown as bar graphsStatistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ****, p<0.0001; **, p<0.01.H) Sections of engrafted skin developed from USP7 KO or control WT cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100μm.
Figure1TEM image and (B) size distribution of pcMNPs; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles.Zeta potentials of bare MNPs, NH2-MNPs and pcMNPs in water. (D) The magnetic response property of bare MNPs, NH2-MNPs and MBs in 30 s.
Figure2RNA binding affinity of pcMNPs analysed by native PAGE;Dispersion stability of pcMNPs in a high-salt solution (50 mM NaCl).
Figure3RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.RT-PCR assays amplifying (A) the ORF1ab region and (B) the N gene in pseudoviral RNA extracted by a manual protocol.
Figure4RT-PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol, and (B) RT-PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol.
Figure5RT-PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT-PCR protocol, and (B) corresponding calibration curve.RT-PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT-PCR protocol, and (D) corresponding calibration curve. The red line is the linear regression fit (R2 = 0.998).
Figure 1(B) Genome browser representation of the SOX2 locus. The promoter is defined as the unmethylated region spanning the SOX2 transcriptional start site in MCF7-control.Genomic distribution of (C) identified ZF-D3A binding sites, and (D) differentially methyl- ated regions (DMRs) between MCF7-control and ZF-D3A +dox.(E) Shown for all DMRs identified between ZF-D3A +dox and MCF7-control is: ∆mCG (difference in the methylation level [ratio of C base calls to total base calls for all CG dinucleotides in region, mCG/CG] between ZF-D3A +dox and MCF7-control), ZF-D3A ChIP-seq signal (normalized ChIP-seq read density, ±1kb flanking DMR center), CpG dinucleotide density (CpG/100bp, ±1kb flanking DMR center), and classification of each DMR as promoter or non-promoter located.(F) DNA methylation level in MCF7-control and ZF-D3A +dox cells flanking (±1 kb, 100 bp bins) the center of ZF-D3A ChIP-seq peaks identified in ZF-D3A +dox.(G) Genomic distribution of unmethylated regions (UMRs) in MCF7-control. (H) DMRs and UMRs with an intersection of ≥1 bp.