EMBO/soda-nel huggingface dataset:
Identification of integrin-linked kinase (ILK) as a critical regulator of VEGFR3 signalling and lymphatic vascular growth (2018)
Figure 4

Figure 4

The lymphatic vascular effect of Ilk deletion strictly depends on β1 integrin.

A, B Bright-field image of an E13.5 Flk1-Cre;Ilk∆/+;Itgb1∆/+ mouse embryo (referred to as 'Control') with a heterozygous deletion of both Ilk and Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jugular lymph sac / primordial thoracic duct (jls/pTD). Scale bars: 500 and 100 µm, respectively.

C, D Bright-field image of an E13.5 Flk1-Cre;Ilk∆/∆;Itgb1∆/+ embryo (referred to as 'ILK & β1 integrin K.O.') with a homozygous deletion of Ilk and heterozygous deletion of Itgb1 in endothelial cells, and a LSM image of a stained cross-section through its jls/pTD. Scale bars: 500 and 100 µm, respectively.

E-H LSM images of cross-sections through the jls/pTD of E13.5 control and ILK & β1 integrin K.O. embryos stained for the proliferation marker phospho-Histone H3. Arrows point to phospho-Histone H3-positive LECs. Scale bars: 20 µm.

I-L LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on stained cross-sections through the jls/pTD of control and ILK & β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1 stained area. Scale bars: 10 µm.

M Number of LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryos.

N LEC proliferation as determined by the number of phospho-Histone H3-positive LECs per jls/pTD section in E13.5 control or ILK & β1 integrin K.O. embryos.

O Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK & β1 integrin K.O. embryos.

Data information: Data are presented as means ± SEM, shown as percentage of control embryos with n = 5 embryos per genotype, unpaired two-tailed Student's t-test.
Identification of integrin-linked kinase (ILK) as a critical regulator of VEGFR3 signalling and lymphatic vascular growth (2018)
Figure 5

Figure 5

ILK controls lymphatic vascular growth and VEGFR3 tyrosine phosphorylation in adult mice.

A-D LSM images of a whole mount stained adult Prox1-CreERT2;Ilk+/+ mouse ear (referred to as 'Adult Control') and Prox1-CreERT2;Ilk∆/∆ mouse ear (referred to as 'Adult ILK K.O.'), with higher magnification images indicated by dashed lines. Scale bars: 500 and 100 µm, respectively.

E-H LSM images of a whole-mount stained adult control and ILK K.O. ear, with higher magnification images indicated by dashed lines. Arrows point to proliferating LECs. Scale bars: 100 and 20 µm, respectively.

I Dermal lymph vessel density as determined by VEGFR3-positive area normalised to the total analysed area of adult control or ILK K.O. mice (n = 4 control and n = 5 ILK K.O. mice), *P = 0.025.

J LEC proliferation as determined by phospho-Histone H3-positive LECs normalised to analysed VEGFR3-positive area in the skin of adult control or ILK K.O. mice (with n = 7 control and n = 5 ILK K.O. mice), *P = 0.004.

K VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K.O. mice (n = 3 mice per genotype), P = 0.062.

L, M LSM images of a whole mount stained adult control and ILK K.O. cornea, with detailed images on their right (L', M'). Arrows point to lymph vessels protruding into the cornea (encircled by a dotted line). Scale bars: 500 µm and 100 µm, respectively.

N Number of lymph vessels in control or ILK K.O. corneas (n = 5 and n = 7 corneas), *P = 0.02.

Data information: Data are presented as means ± SEM, shown as percentage of control mice, unpaired two-tailed Student's t-test.
Identification of integrin-linked kinase (ILK) as a critical regulator of VEGFR3 signalling and lymphatic vascular growth (2018)
Figure 6

Figure 6

ILK controls lymphatic vascular growth in the heart after myocardial infarction.

A, B LSM images of cross-sections through the heart of an adult Prox1-CreERT2;Ilk+/+ mouse (referred to as 'Adult Control') and Prox1-CreERT2;Ilk∆/∆ mouse (referred to as 'Adult ILK K.O.') showing the outer lateral region of the left ventricle (LV). Scale bars: 100 µm.

C, D Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively (n = 4 control and n = 5 ILK K.O. mice).

E, F LSM images of cross-sections through the heart of an adult control and ILK K.O. mouse four weeks after myocardial ischemia and reperfusion (MI/R), showing the outer lateral region of the LV. Scale bars: 100 µm.

G, H Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively. (n = 5 control and n = 7 ILK K.O. mice), P = 0.051 (cardiac lymph vessel density in adult control versus adult ILK K.O.), *P = 0.025 (cardiac blood vessel density in adult control versus adult ILK K.O.).

Data information: Data are presented as means ± SEM, shown as percentage of control mice, unpaired two-tailed Student's t-test.
Identification of integrin-linked kinase (ILK) as a critical regulator of VEGFR3 signalling and lymphatic vascular growth (2018)
Figure 7

Figure 7

ILK controls proliferation, VEGFR3 signalling, and VEGFR3-β1 integrin interactions in human LECs.

A, B Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 µm.

C LEC proliferation as determined by the number of BrdU-positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 3 independent transfections per siRNA), *P = 0.032 (control versus ILK-1), *P = 0.005 (control versus ILK-2), *P = 0.0003 (control versus ILK-3).

D VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 4 (control siRNA, ILK-1 siRNA, and ILK-3 siRNA), or n = 8 (ILK-2 siRNA) independent transfections per siRNA), *P = 0.0001 (control versus each siRNA).

E, F LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 µm.

G Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs (n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK-1), *P = 0.024 (control versus ILK-2), *P = 0.001 (control versus ILK-3).

Data information: Data are presented as means ± SEM, shown as percentage of control siRNA, one-way ANOVA with Dunnett's multiple comparisons test.
Identification of integrin-linked kinase (ILK) as a critical regulator of VEGFR3 signalling and lymphatic vascular growth (2018)
Figure 8

Figure 8

Mechanically stretched human LECs have more VEGFR3-β1 integrin and less ILK-β1 integrin interactions.

A-D LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 µm.

E Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (-) mechanical stretch (n = 6 independent stretch chambers), *P = 0.039.

F Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 minutes, and used for immunoprecipitation (IP) of HA-tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates.

G Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (-) mechanical stretch; normalised to the respective amount of HA-tagged β1 integrin (n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers), *P = 0.0007.

Data information: Data are presented as means ± SEM, unpaired two-tailed Student's t-test.

Source data are available for this figure.
Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay (2022)
Figure 1

Figure 1 - UPF3A overexpression does not affect NMD

A Schematic representation of the bridge between UPF1 and the EJC during NMD. Binding of UPF3A instead of the stronger bound UPF3B is discussed to either activate or repress NMD.

B Western blot analyses after induced expression of FLAG-tagged UPF3A in WT HEK293 and HeLa cells (n=1). Tubulin serves as control. The asterisk indicates unspecific bands.

C Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR (ISAR) using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.

D Volcano plot showing the differential gene expression analyses from the RNA-Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A. The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. OE = overexpression.

E Read coverage of SMG5 from WT HEK293 and HeLa RNA-seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer (IGV) snapshot. Differential gene expression (from DESeq2) is indicated as log2 fold change (log2FC) on the right. Schematic representation of the protein coding transcript below.
Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay (2022)
Figure 2

Figure 2 - UPF3A KOs show light NMD-independent transcriptome alterations

A Western blot analysis of WT and UPF3A KO cells (clones 4, 14 and 20) with the indicated siRNA treatments (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.

B Quantitative RT-PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC) (n=3 for RSRC2 and SRSF2, n=4 for ZFAS1).

C Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.

D,E Volcano plots showing the differential gene expression analyses from the indicated RNA-Seq datasets (D UPF3A KO clone 14, E UPF3A KO clone 20). The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method

F nVenn Diagram showing the overlap of up- or downregulated genes in the UPF3A KO cell lines 14 and 20. Log2 fold change <1 (downregulated) or >1 (upregulated) and adjusted p-value (padj) < 0.05. DGE = Differential Gene Expression.
Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay (2022)
Figure 3

Figure 3 - Loss of UPF3B does not affect NMD efficiency, only in combination with KD of UPF3A

A Western blot analysis of WT and UPF3B KO cells (clones 90 and 91) combined with the indicated knockdowns (n=1). UPF3A and UPF3B (AK-141) protein levels were detected, Tubulin serves as control. Protein levels of UPF3A were quantified, normalized to tubulin expression, and shown as datapoints and mean. Fold-changes of relevant conditions are shown. The asterisk indicates unspecific bands.

B Quantitative RT-PCR of the indicated cell lines with the indicated knockdowns. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC, n=3).

C Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.

D,E Volcano plots showing the differential transcript usage (via IsoformSwitchAnalyzeR) in various RNA-Seq data. Isoforms containing GENCODE (release 33) annotated PTC (red, TRUE), regular stop codons (blue, FALSE) or having no annotated open reading frame (grey, NA) are indicated. The change in isoform fraction (dIF) is plotted against the -log10 adjusted p-value (adj. p-value). Density plots show the distribution of filtered isoforms in respect to the dIF, cutoffs were |dIF| > 0.1 and adj. p-value < 0.05. P-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method.
Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay (2022)
Figure 4

Figure 4 - KO of both UPF3 paralogs results in strongly impaired NMD

A Western blot analysis of WT, UPF3B KO and UPF3A-UPF3B double KO cells (clones 1 and 2) (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.

B Schematic depiction of the insertion/deletion in the UPF3A open reading frame resulting in the additional UPF3A KO in the UPF3B KO clone 90 generating UPF3 dKO clones.

C Quantitative RT-PCR of the indicated samples with the indicated KDs. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).

D Schematic overview of the globin reporter constructs and their functional elements.

E Northern blot analysis of globin reporter and xrFrag. Ethidium bromide stained 28S and 18S rRNAs are shown as controls. Quantification results are shown as data points and mean (n=3).
Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay (2022)
Figure 6

Figure 6 - Interaction of UPF3A and UPF3B with the EJC is dispensable to elicit NMD

A-C Volcano plots of label free mass spectrometry-based analysis of the interaction partners of UPF2 in WT cells treated with control siRNAs and the UPF3B KO clone 90 and dKO clone 1 both treated with siRNAs targeting UPF3B (n = 4 biologically independent samples). (A) FLAG-UPF2 in WT against FLAG-GST control in WT cells, (B) UPF2 in 3B KO cells against FLAG control in WT cells, (C) UPF2 in dKO cells against FLAG control in WT cells. Points labeled in purple indicate NMD factors; points labeled in turquoise indicate EJC components. Cut offs: Log2 fold change ≥ 0

D Schematic representation of the UPF3B protein domains and the respective functions. Below are the mutated rescue constructs and their respective abstract placeholders. RRM = RNA recognition motif, EBM = EJC binding motif.

E Representative Western blot after FLAG co-immunoprecipitation (IP) of FLAG-tagged GST (control) or UPF3B WT and single mutant constructs in UPF3B KO cells (n = 3).

F Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3B rescue construct shown in (D). Rescue construct protein levels were detected with anti-FLAG and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1).

G Quantitative RT-PCR of the samples from (F). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).
Human UPF3A and UPF3B enable fault-tolerant activation of nonsense-mediated mRNA decay (2022)
Figure 7

Figure 7 - WT UPF3A can rescue NMD in full extent. Deletion on exon 4 disrupts functionality in both paralogs

A Schematic representation of the UPF3A and UPF3B protein domains. Below are the respective mutated rescue constructs.

B Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3A and UPF3B rescue construct shown in (A). Rescue construct protein levels were detected with anti-FLAG, anti-UPF3A and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1). The asterisk indicates unspecific bands.

C Quantitative RT-PCR of the samples from (B). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC) (n=3).
Brassinosteroid signals cooperate with microtubule severing to control filament elongation (2022)
Figure 2

Figure 2 The microtubule-severing defective mutants display abnormal microtubule organization in the stamen filament cells

Comparison of microtubule organization in the stamen filament cells of wild type, lue1, and ktn80.1234. Scale bar, 10 μm.

Microtubule morphology distribution in wild type and katanin mutants. Seven stamen filaments from different plants were analyzed in wild type, five stamen filaments from different plants were analyzed in lue1 and ktn80.1234, respectively. Error bars indicate SD. Statistically significant differences between groups were tested using One-way ANOVA, **P < 0.01. Wild type versus lue1 in parallel microtubule arrays, **P < 0.0001; wild type versus ktn80.1234 in parallel microtubule arrays, **P < 0.0001; wild type versus lue1 in oblique microtubule arrays, **P < 0.0001; wild type versus ktn80.1234 in oblique microtubule arrays, **P < 0.0001; wild type versus lue1 in disordered microtubule arrays, **P < 0.0001; wild type versus ktn80.1234 in disordered microtubule arrays, **P < 0.0001.

Microtubule organization in wild type stamen filament cells. Scale bar, 10 μm.

(D-F) Microtubule organization in the stamen filament cells of lue1 and ktn80.1234. Arrows indicate microtubule crossovers. Dotted lines indicate abnormal microtubules. Scale bar, 10 μm.

(G) Microtubule density in wild type and katanin mutants. Six cells from different plants were analyzed in wild type, five cells from different plants were analyzed in lue1 and ktn80.1234, respectively. Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus lue1, **P = 0.0031; wild type versus ktn80.1234, **P = 0.0098.

(H) S2 values in wild type and katanin mutants. Five cells from different plants were analyzed in wild type, five cells from different plants were analyzed in lue1 and ktn80.1234, respectively. Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus lue1, **P = 0.0033; wild type versus ktn80.1234, **P = 0.0006.
Brassinosteroid signals cooperate with microtubule severing to control filament elongation (2022)
Figure 3

Figure 3 The ktn80.1234 quadruple mutant lacks proper microtubule severing and disrupts the recruitment of KTN1 in the stamen filament cells

The recruitment of GFP-labeled KTN1 (pseudo-colored in red) to the microtubules (mCherry-TUB6; pseudo-colored in green) is completely disrupted in ktn80.1234 filament cells compared with wild type. Scale bar, 5 μm.

Severing frequency (severing events per 100 μm2 per second) from all severing events. 165 severing events were detected in area of 1087 μm2 from three cells in wild type over 5 minutes. 15 severing events were detected in area of 1187 μm2 from three cells in ktn80.1234 over 5 minutes. Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus ktn80.1234, **P < 0.0001.

Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites in wild type filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at microtubule crossover sites. Asterisks indicate the location at which a microtubule has been severed at a crossover site. Scale bar, 5 μm.

Time-lapse images showing that KTN1 cannot be recruited to crossover sites and no severing was observed in ktn80.1234 filament cells. Yellow open arrows indicate microtubule crossovers, which remain stable for a long time. Scale bar, 5 μm.
Brassinosteroid signals cooperate with microtubule severing to control filament elongation (2022)
Figure 4

Figure 4 The qui-2 has short filaments and short filament cells

Comparison of the filament and stigma phenotypes in wild type and qui-2. Scale bars, 0.2 mm.

Gynoecium length in wild type and qui-2 at anthesis. N = 81 gynoeciums from 9 wild type plants, n = 42 gynoeciums from 15 qui-2 plants. Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus qui-2, **P < 0.0001.

Stamen length in wild type and qui-2 at anthesis. N = 162 stamens from 9 wild type plants, n = 84 stamens from 15 qui-2 plants. Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus qui-2, **P < 0.0001.

Ratio of the stamen to stigma length in wild type and qui-2 at anthesis. N = 81 stamen-stigma pairs from 9 wild type plants, n = 42 stamen-stigma pairs from 15 qui-2 plants. Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus qui-2, **P < 0.0001.

Comparison of the morphologies of the stamen filament cells in wild type and qui-2. Scale bars, 10 μm.

Stamen filament cell length in wild type and qui-2. N = 61 cells from 5 wild type plants, n = 62 cells from 15 qui-2 plants. Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus qui-2, **P < 0.0001.

Stamen filament cell width in wild type and qui-2. N = 61 cells from 5 wild type plants, n = 62 cells from 15 qui-2 plants. Error bars indicate SD. Student's t test, *P < 0.05. Wild type versus qui-2, *P = 0.0156.

Ratio of filament cell width/length in wild type and qui-2. N = 61 cells from 5 wild type plants, n = 62 cells from 15 qui-2 plants. Error bars indicate SD. Student's t test, **P < 0.01. Wild type versus qui-2, **P < 0.0001.