EMBO/soda-nel huggingface dataset:
ERp18 regulates activation of ATF6α during unfolded protein response (2019)
Figure 5

Figure 5: ERp18 Knockout Downregulates the Transcription and Translation of ATF6α Target Genes during ER stress

CRISPR/Cas9-based knockout of ERp18 in HEK293T cells expressing ATF6α. ERp18 levels in control (WT) and knockout (KO) cells were detected with an antibody to ERp18.

qPCR analysis of UPR target genes in the absence of ER stress. Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type. Error bars represent ± standard deviation for three independent experiments.

qPCR analysis of UPR target genes following ER stress. Wild type and ERp18 knockout HEK293T cells were treated with 1 µM TG for approximately 16 h prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type controls. Error bars represent ± standard deviation for three independent experiments.

Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).

Normalised BiP levels from (D ) were quantified. Error bars represent ± standard deviation for three independent experiments.

Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).

Normalised ERp72 levels from (F) were quantified. Error bars represent ± standard deviation for three independent experiments.
ERp18 regulates activation of ATF6α during unfolded protein response (2019)
Figure 6

Figure 6: Deletion of ERp18 Regulates Cleavage of ATF6α

A, B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times. Cells were then subject to differential centrifugation to obtain membrane (A) and nuclear fractions (B) which were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of full-length ATF6α (ATF6-FL), cleaved membrane-associated ATF6α (ATF6-P) and nuclear translocated S2P cleaved ATF6α (ATF6-N) are as indicated. HDAC2 was used as a nuclear marker. Data shown is representative of two independent experiments.

(C) ATF6α was immunoisolated from wild type or ERp18 KO cells expressing ATF6α. Samples were separated by SDS-PAGE and ATF6α was detected by western blotting. HC indicates immunoglobulin heavy chains.

(D, E) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α, either untreated (-) or treated (+) with 30 µM PF429242 (S1P inhibitor) prior to and following induction of ER stress with 10 mM DTT. The positions of ATF6-P, ATF6-M and ATF6-N are as indicated. The unprocessed ER-localised ATF6α (designated ER) and the O-linked glycan modified Golgi-translocated ATF6α (designated G) are indicated. HDAC2 was included as a nuclear marker. The blots in (D, E) confirm that ATF6-P is produced independently of S1P and also the absence of ATF6α processing to ATF6-N in the presence of the S1P inhibitor.
ERp18 regulates activation of ATF6α during unfolded protein response (2019)
Figure 7

Figure 7: ATF6α Cleavage Occurs in a post-ER compartment

HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor. Cell lysates were prepared, separated by SDS-PAGE and then probed with anti-ATF6α. Blot reveals the presence of O-linked glycan modified Golgi-translocated ATF6 (designated G), which is absent for the D564G mutant, confirming a lack of ER to Golgi trafficking.

Wild type and ERp18 KO cell lines stably expressing ATF6α D564G were either left untreated (-) or treated (+) with 5 µg/ml brefeldin A for1 h prior to cell lysis. The anti-ATF6α western blot of membrane fractions reveals proteolytic processing by S1P to produce ATF6-M, only in the brefeldin A treated cells. Nuclear fractions confirm the presence of S2P cleavage product, ATF6-N following brefeldin A treatment. Anti-HDAC2 was used as a loading control for the nuclear fraction.
ERp18 regulates activation of ATF6α during unfolded protein response (2019)
Figure 8

Figure 8: Deletion of ERp18 Promotes ER to Golgi Trafficking of ATF6α

A-C Wild type cells stably expressing ATF6α (A) or ERp18 KO cells stably expressing ATF6α (B), and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 (RV cells) (C), were pre-treated with 30 µM PF429242 (S1P inhibitor) for 1 h prior to induction of ER stress with 10 mM DTT as indicated.

D-F Wild type cells transiently expressing ATF6 C467A (D), ATF6 C618A (E), or ATF6 DM (F) were treated as above. Whole cell lysates were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of unprocessed ER-localised ATF6α (ER) and the O-linked glycan modified Golgi-translocated ATF6α (Gol) are indicated as is ATF6-P.

G The percentage of Golgi-ATF6α was quantified, the error bars represent ± standard deviation for at least three independent experiments.
Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data (2019)
Figure 1

Figure 1. UACC257 and A2058 melanoma cells are a model system to globally study EDNRB signalling.

A Kinetics of intracellular Ca2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB-KO) were seeded on 96 well plates, loaded with Fluo-4 dye and stimulated with 100 nM EDN or PBS. Fluo-4 fluorescence was monitored over ten minutes (n = 6). Representative example of three independent experiments.

B Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2-90 min. AKT and CREB phosphorylation and expression was evaluated by Western blot. Representative example of three independent experiments.

C Experiment design for the UACC257 phosphoproteomic study, showing time points, cell line derivatives, replicate structure and GIST composition. The GIST was generated by pooling all heavy SILAC plates (blue) and spiked into all UACC257 (red) and UACC257 EDNRB-KO (green) samples. The A2058 cell line was also analysed in biological triplicates and a GIST but without EDNRB-KO cells.

D High reproducibility in the UACC257 and A2058 datasets. CV distributions for triplicates were calculated and are provided in Figure EV1D. All CV values were grouped according to cell line to represent dataset specific CV distributions. Numbers and lines indicate the median.
Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data (2019)
Figure 2

Figure 2. EDN elicits a robust phosphorylation response, which is partly shared between melanoma cell lines.

A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups and shading is mapped to time.

B Overlap between UACC257 and A2058 datasets. From top to bottom: Identified proteins, identified phosphopeptides, EDN regulated phosphopeptides at q < 0.1, and q < 0.1 & FC > 1.5 up or down.

C Comparison of the kinetics of the 242 shared target phosphopeptides. Log2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.
Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data (2019)
Figure 4

Figure 4. Target phosphopeptides show six distinct response kinetics, which are shared between cell lines and are associates with similar sets of kinases.

A,B EDN target phosphopeptides for (A) UACC257 and (B) A2058 were segregated into six clusters each, using c-means clustering (MFuzz). Each line represents one phosphopeptide and cluster membership is indicated by colour. Most likely kinases for each phosphosite were predicted using NetworKIN and enrichment of kinase substrates was calculated for each cluster. Enriched kinases are indicated above each cluster. Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster. Detailed information on the kinase enrichment is provided in Appendix Table S1.

C Average MS1 intensity z-score kinetics for 217 predicted CaMKII-α sites that are shared between both cell lines. For comparison, average (n = 6) calcium abundance kinetics from Fig. 1A are shown.
Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data (2019)
Figure 6

Figure 6. Validation of kinase predictions using marker substrates.

Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed. UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min (5 min for CaMKII). Ten μg protein from RIPA lysates were analysed by Western blot. Kinase marker substrate phosphorylations and corresponding loading controls are shown. For each kinase, two independent replicates were performed and are shown. Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band identity (extended versions with inhibitors are shown in Figure EV4).
Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data (2019)
Figure 7

Figure 7. PKC, JNK, AMPK and AKT mediate EDN induced cell migration.

The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay. Scratches were made in UACC257 monolayers. Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0.1 μM or 1 μM in triplicates.

A Example pictures showing the effect of EDN on migration. Cell free areas are delimited by blue lines. Migration was quantified as the change in cell free area between 0 h and 48 h.

B The four inhibitors that were effective at 1 µM are shown in detail, i.e. every point corresponds to one well.

C Abundance of E-Cadherin (normalized to GAPDH and Actin) was assessed by LC-SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM. Only the single inhibitor which could reverse EDN induced E-Cadherin repression is shown. The full SRM dataset is provided in Appendix Figure S3.

* p < 0.05 two-sided unpaired t-test.

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Nucleotide Signaling Pathway Convergence in a cAMP-sensing bacterial c-di-GMP Phosphodiesterase (2019)
Figure 1

Figure 1. Bd1971 Expression and Localization During the Bdellovibrio Lifecycle

(A) Reverse Transcriptase PCR showing that bd1971, and control gene dnaK, are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus. RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E. coli cells. Primers were designed to anneal specifically to the gene of interest. L = 100bp DNA ladder, AP = Attack Phase cells, 15-45 = 15-45 minutes respectively since infection, 1h-4h = hours respectively since infection. Ec = E. coli strain S17-1 RNA (negative control: no Bdellovibrio RNA); NT = control with no template RNA; gen = B. bacteriovorus HD100 genomic DNA (positive control). The cartoon above represents each stage in the predatory cycle.

(B) Epifluorescent images of E. coli invaded by B. bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection. Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images (see methods) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation. Fluorescence images and merges with phase contrast images are shown. Scale bars are 2 µm. Images are representative of three independent experiments.
Nucleotide Signaling Pathway Convergence in a cAMP-sensing bacterial c-di-GMP Phosphodiesterase (2019)
Figure 5

Figure 5. Phosphodiesterase activity of Bd1971 is stimulated by the allosteric effectors cAMP and cGMP.

(A) Apo-Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations (2.5 mM) of cAMP, cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP (5 mM) was measured spectrophotometrically. Values are the average of three repeats, error bars represent standard deviation.

(B) HPLC traces of reaction products generated from 100 µM c-di-GMP incubated with apo-Bd1971 (Control), or protein supplemented with either 100 µM cAMP or cGMP. Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure.
Nucleotide Signaling Pathway Convergence in a cAMP-sensing bacterial c-di-GMP Phosphodiesterase (2019)
Figure 8

Figure 8. Binding of cAMP and cGMP to the cNMP domain of Bd1971 measured by isothermal calorimetry.

A, B ITC titration curves and relative heats of binding for (A) cAMP and (B) cGMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment. Data was analysed with Origin software and thermodynamic parameters associated with these binding experiments are presented in Table 2. Results presented are representative of two independent experiments.
Nucleotide Signaling Pathway Convergence in a cAMP-sensing bacterial c-di-GMP Phosphodiesterase (2019)
Figure 11

Figure 11. Bacterial two hybrid (BTH) assays showing interactions between Bd1971 and c-di-GMP synthesizing or binding proteins.

(A) Spot tests of co-transformants of either Bd0367 (DgcA; GGDEF protein) response regulator domain or Bd3125 (CdgA; a degenerate GGDEF protein likely involved in c-di-GMP binding and signalling) with full length Bd1971, or the N-terminal (1-145), or C-terminal (145-400) portions of Bd1971 in either pUT18c or pKT25 as indicated. Positive control was co-transformants with pUT18c-zip and pKT25-zip, negative control was the empty vectors. The co-transformants were plated on LB-X-Gal medium with positive interaction resulting in blue colouration, which can be seen for all of the interactions in both plasmid combinations, but not for the negative control. Images are representative of at least 4 independent experiments.

(B) Confirmation of BTH interactions by β-Galactosidase activity assays. The interaction is significantly stronger between Bd3125 and the N-terminal domain of Bd1971 than the interaction with the C-terminus of Bd1971. The response regulator domain of Bd0367 showed significant interaction with Bd1971, but the full length Bd0367 did not. *p>0.05 by Student's t-test N/S- not significant. Data are from at least three independent experiments, error bars are the standard error of the mean

Expanded View Figure Legend
Overcoming Resistance to Anabolic SARM Therapy in Experimental Cancer Cachexia with a HDAC Inhibitor (2019)
Figure 5

Figure 5. A) Gene expression of multiple cachexia-associated markers in gastrocnemius muscles of individual animals from Study 1 (n=5 for tumor-bearing groups; n=6 for tumor-free groups). Expression was determined by qRT-PCR and presented as described in the Materials and Methods (Geometric Mean ± Geometric STD). B) Androgen receptor (AR) mRNA expression in gastrocnemius muscles from Study 1. Statistics: p values provided in Appendix Table S5A-B. Dunnett's multiple comparison test. CEBPδ, n=4, insufficient sample to analyze all tumor-bearing GTx-024-treated animals.
Overcoming Resistance to Anabolic SARM Therapy in Experimental Cancer Cachexia with a HDAC Inhibitor (2019)
Figure 6

Figure 6. A) ELISA analysis of serum IL-6 levels in terminal samples from Study 2. Tumor-free groups (n=6), tumor-bearing receiving Vehicle (n=7), GTx-024 (n=10), AR-42 (n=8) or Combination (n=9). ND, not detected. B and C) Phospho(p)STAT3 (Y705)/total (t)STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 (B) and Study 3 (C). Black circle - tumor-free, blue square - tumor-free/GTx-024, black triangle - tumor-bearing, blue diamond - tumor/GTx-024, red triangle - tumor/AR-42, green circle - tumor/combo. D) Spleen weights normalized to tumor-corrected terminal body weights of mice from Study 2. Tumor-free groups (n=6), tumor-bearing Vehicle (n=7), tumor-bearing receiving GTx-024 (n=10), AR-42 (n=9) or Combination (n=9). E) ELISA analysis of pSTAT3(Y705)/tSTAT3 within C-26 tumors from Study 2. Tumor-bearing Vehicle-treated (n=7), tumor-bearing receiving GTx-024, AR-42 or Combination (n=8). Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p vales provided in Appendix Table S6, one-way ANOVA followed by Tukey's multiple comparison test (mean±SD). Panel E, one sample from each of the treatment groups was not available for analyses. BW, body weight.
Overcoming Resistance to Anabolic SARM Therapy in Experimental Cancer Cachexia with a HDAC Inhibitor (2019)
Figure 7

Figure 7. A) Effect of GTx-024 and AR-42 monotherapies on cachexia-related differentially regulated genes (DEGs) from RNA-seq analyses of Study 1 gastrocnemius muscles. All three panels consist of individual genes plotted with respect to their log2 fold change and -log10 Benjamini-Hochburg adjusted p-values from the comparison of cachexia vs tumor-free controls. Colors of the points reflect the DEG status of each gene for the given comparison. B) Results from transcription factor pathway-focused GSEA of tumor-bearing (cachexia) versus tumor-free control transcriptomes. STAT3 and ATF-1 gene sets used for subsequent combined analyses are hatched. C) Significance values from GSEA using combined STAT3 gene sets identified in B. Each treatment group is compared to tumor-bearing control (cachexia) transcriptomes. D) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data. Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). E) Results of GSEA using combined ATF-1 gene sets identified in B across treatment groups versus tumor-bearing control (cachexia) transcriptomes. F) Heat map of DEGs within the combined ATF-1 gene sets (mean z score). Treatment groups are as in D. G) mRNA expression of mediators of IL-6 signaling upstream of STAT3. Data presented as mean±SD of per animal log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6). *p<0.1, **p<0.05, ***p<0.01, ****p<0.001 based on Benjamini-Hochburg adjusted p-values from DESeq2. Exact p values are provided in Appendix Table S7.
Overcoming Resistance to Anabolic SARM Therapy in Experimental Cancer Cachexia with a HDAC Inhibitor (2019)
Figure 8

Figure 8. A) Volcano plot from RNA-seq analyses of Study 1 gastrocnemius muscles for tumor-bearing combination-treated mice versus tumor-bearing controls. Genes not differentially expressed in this comparison are indicated with blue. The remaining genes (red and green) are DEGs in the combination-treated versus tumor-bearing control comparison. The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR-42-treated tumor-bearing mice versus tumor-bearing controls, suggesting these genes are responsive to only the combination therapy. Log2-transformed fold change (FC) in expression is plotted on the x-axis and -log10 transformed Benjamini-Hochburg adjusted p-values are plotted on the y-axis. B) Significance values from transcription factor pathway-focused GSEA of GTx-024-treated tumor-free versus tumor-free control transcriptomes. *FDR< 1e-5 was determined for the CTNNB1 gene set, and was set to 1e-5 for the plot. C) Significance values from GSEA using combined CTNNB1 gene sets. Each treatment group was compared to tumor-free control transcriptomes. *FDR < 1e-5 determined, set to 1e-5 for plot. D) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor-free control comparisons. GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). E) mRNA expression of WNT effectors upstream of β-catenin. Data are presented as mean±SD of log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6). *p<0.1, **p<0.05, ***p<0.01, ****p<0.001 based on Benjamini-Hochburg adjusted p-values from DESeq2. Exact p-values provided in Appendix Table S8.
Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes (2021)
Figure 2

Figure 2. AP2-O3 null female gametocyte fails to develop fully formed female gamete.

In vitro exflagellation rate of male gametocytes indicating male gamete formation. mean ± SEM from three experiments. Two-tailed unpaired Student's t-test.

Western blot of P28 protein expression in the non-activated gametocyte (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control. The numbers are the relative intensities of P28 band normalized to the BiP band in the blot.

Co-staining of P28 and GAP45 (IMC protein) in female gametes (P28+ only) and zygotes (P28+/GAP45+) of the 17XNL, Δap2-o3 and Δcdpk4 strains. Percentages of female gametes and zygotes were indicated respectively. n is the number of cells counted. Scale bars = 5 μm. Note: the signal of P28 and GAP45 is reduced similarly in all Δap2-o3 female gametes and zygotes compared to that of 17XNL.

Time-course analysis of zygote to ookinete development in vitro. Upper diagrams indicate morphological changes from zygote to ookinete. Scale bars = 5 μm.

Quantitative counting of different stages during parasite development in D. Data are gathered from 3 independent experiments and presented as mean ± SEM.

Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, Δap2-o3, Δmap2, or Δnek4 strain alone as well as mixtures of Δmap2/Δnek4, Δap2-o3/Δmap2, and Δap2-o3/Δnek4. Δnek4 and Δmap2 are female and male gamete-defect parasites, respectively. n is the number of mosquitoes dissected, Mann-Whitney test applied, two experiments repeated.

Schematic of the defects caused by AP2-O3 deficiency during female gametocyte to ookinete development.