EMBO/soda-nel huggingface dataset:
Neuron-specific translational control shift ensures proteostatic resilience during ER stress (2022)
Figure 5

Figure 5: The ER stress-induced ATF4 response in PERK-deficient neurons is fully p-eIF2α-dependent

A-F Protein synthesis (A-C, N=3) and ATF4 expression (D-F, N=5) in WT and Perk KO neurons overexpressing eIF2αWT or phospho-deficient eIF2αS51A ± 20-24 hours of TM-induced ER stress. Representative images obtained by (A) confocal and (D) high-content microscopy. Dendrites (MAP2, white/cyan), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (B) and ATF4 response (E). Data are normalised to WT eIF2αWT. Quantification showing the percentage change in the ER stress-induced protein synthesis (C) and ATF4 (F) response induced by eIF2αS51A compared to eIF2αWT. (C) Calculated from data in (B): [(eIF2αS51A-eIF2αWT)/eIF2αWT] *100. (F) Calculated from data in (E): [(eIF2αS51A-eIF2αWT)/eIF2αWT]*-100.

G-L Expression of p-eIF2α sensitive (5'ATF4-uORFWT) and p-eIf2α insensitive (5'ATF4-uORFMut) EYFP translational reporters in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=3). (G, H) Schematic depiction of the reporters. (I, K) Representative confocal images. Dendrites (MAP2, cyan), nuclei ((Δ)Cre, red), endogenous ATF4 (magenta), ATF4 reporter (green). Scale bar: 10 μm. (J, L) Quantification of EYFP intensity, normalised to untreated WT.

Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E, J, L); Nested t-test (C, F). Source data is available online.
Neuron-specific translational control shift ensures proteostatic resilience during ER stress (2022)
Figure 6

Figure 6: ER stress-induced reduction of protein synthesis in PERK-deficient neurons is partially eIF2-independent

A-F Protein synthesis and ATF4 expression in ISRIB-treated WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=4). (A, D) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm.

Quantification of the ER stress-induced protein synthesis (B) and ATF4 (E) response. Data are normalised to WT without ISRIB. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress-induced protein synthesis (C) and ATF4 (F) response induced by ISRIB. (C) Calculated from data in (B): [(ISRIB+-ISRIB-)/ISRIB-]*100. (F) Calculated from data in (E): [(ISRIB+-ISRIB-)/ISRIB-]*-100.

G-M Protein synthesis and ATF4 expression in 2b5he and 2b5ho astrocytes and neurons ± 20-24 hours of TM-induced ER stress in the presence or absence of PERK-i (Pi) (N=3). (G, I, K, M) Representative images obtained by high-content microscopy. Astrocytic soma (Phalloidin, white), dendrites (MAP2, white), nuclei (DAPI, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. (H, L) Quantification of puromycin intensity as measure for de novo protein synthesis and (J, N) absolute nuclear ATF4 intensity. Data are normalised to untreated WT.

Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F); Three-way ANOVA with Tukey's post hoc test (H, J, L, N). Source data is available online.
Neuron-specific translational control shift ensures proteostatic resilience during ER stress (2022)
Figure 7

Figure 7: PERK-deficient neurons activate angiogenin-mediated protein synthesis reduction

A Differentially expressed genes (DEGs) involved in oxidative stress related processes (Fig. 4A), comparing TM-treated Perk KO and WT neurons. Blue arrow: Ang. See also Appendix Figure S2.

B, C Ang mRNA level in WT and Perk KO neurons and astrocytes ± 20-24 hours of TM-induced ER stress, determined by mRNA-seq (N=3). FPMK: Fragments Per Kilobase of transcript per Million.

D, E Representative WB and quantification of ANG protein level (N=3).

F, G tRNA unfolding in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=4). (F) Representative images obtained by high-content microscopy of 1-methyladenosine (m1A) immunofluorescence to determine tRNA unfolding. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), unfolded tRNA (m1A, green). Scale bar: 25 μm. (G) Quantification of m1A intensity, normalised to untreated WT.

H-M Abundance of small RNAs in WT and Perk KO astrocytes (N=3) and neurons (N=6) ± 20-24 hours of TM-induced ER stress, determined by high-resolution automated electrophoresis (H, K) Representative gel image. Electropherogram (I, L) and zoom (J, M) showing quantified fluorescence units (FU) of small RNAs, size ranges of tRNAs and tsncRNAs are indicated. Shown is the mean of the biological replicates.

N-R Protein synthesis and ATF4 expression in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress in the presence or absence of ANG-I (N=3). (N) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesis of proteins (Puromycin, green). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (O) and ATF4 response (Q). Data are normalised to WT without ANG-i. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress-induced protein synthesis (P) and ATF4 response (R) induced by ANG-i. (P) Calculated from data in (O): [(ANG-i+-ANG-i-)/ANG-i-]*100. (R) Calculated from data in (Q): [(ANG-i+-ANG-i-)/ANG-i-]*-100.

Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, C, E, G, O, Q); Nested t-test (P, R). Source data is available online.

Table 1: Primers and probes used for qPCR

Extended view Figure Legends
The energy sensing LKB1–AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis (2007)
figf1

(A, B) Two-dimensional tryptic mapping of YFP–p27, YFP–p27T198A and a mixture of both (A). Phosphoamino-acid analysis of spots a and b (B). The migration of phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) is marked with circles in B. The asterisk indicates the origin of migration. (C–E) MCF-7 transfected with YFP–p27 or YFP–p27T198A mutant (C, D) or cotransfected with YFP–p27 and an empty RFP vector in a 5:1 (YFP–p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E). (F) Western blots of protein extracts from MCF-7 transfected with YFP–p27, YFP–p27T198A or YFP–p27T198D mutants following incubation with cycloheximide (CHX) for the indicated times. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5. The scale bars represent 100 μm in D and E.
The energy sensing LKB1–AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis (2007)
figf2

(a) Crystal violet staining of MCF-7 cell transfected with YFP (C1), YFP–p27, YFP–p27T198A or YFP–p27T198D and selected with G418 for three weeks. Data are presented as mean ± s.d. (n = 3). (b) Flow cytometry analysis of MCF-7 cells transfected with YFP (C1) or YFP–p27, YFP–p27T198A and YFP–p27T198D for 48 h. Data are presented as mean ± s.d. (n = 3). (c) GFP–LC3 U2OS cells 48 h post-transfection with RFP, RFP–p27, RFP–p27T198A or RFP–p27T198D. Data represent results of three independent experiments, from which a total of ∼200 cells were counted (s.d., 10∼15%). The scale bars represent 50 μm in c.
The energy sensing LKB1–AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis (2007)
figf3

(a, b) MCF-7 cells were cultured for 24 h following siRNA with or without FBS before western analysis (a) and cell-viability assays (b). (c) MCF-7 cells were transfected with vector alone (C1), YFP–p27 (WT), or the siRNA-resistant constructs WT-R and T198A-R 24 h before RNA interference (RNAi). PARP cleavage was determined by western blotting and densitometric scanning. Data represent three independent experiments (mean ± s.d.). (d) Western blots of lysates of MCF-7 cells transfected with siRNA before serum-starvation or treatment with LY294002 (LY, 10 μM) or rapamycin (Rap, 100 nM) for 48 h with or without methylpyruvate (MP, 10 mM). (e) MCF-7 cells treated with or without LY294002 for 48 h following RNAi. p27 siRNA pool-1, siRNA1, 2 and 3; arrows, membrane blebbing; triangles, apoptotic bodies. The scale bar represents 50 μm. (f) Western blots of protein extracts from MCF-7 cultured with (+) or without (−) glucose for 24 h following RNAi. Fr, fragment; percentage PARP cleavage = 100 × PARPFr / (PARP + PARPFr).
The energy sensing LKB1–AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis (2007)
figf4

(a) Western blots of lysates from the indicated cell lines. (b) Pearson correlation analysis of p27 and LKB1 protein levels quantified by reverse phase protein array assays in 74 randomly selected primary breast tumors (MDACC). (c) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild-type LKB1 cultured with or without glucose for 24 h. (d–f) Western blots of protein extracts from MEFs treated with AICAR (d and e), from 3T3L1 cells cultured with or without glucose for 30 min (f). (g) Western blot analysis of 3T3L1 cells 72 h post-transfection RNAi with or without glucose starvation for 1 h before analysis. Densitometric quantification of western blots is shown. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.
The energy sensing LKB1–AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis (2007)
figf5

(A) FACS analysis of TUNEL positive p27+/+ and p27−/− cells treated with AICAR for 20 h. Data represent mean ± s.d. of three independent experiments. (B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a–f and 0.5 μm in a′–f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).
Stromal cell diversity associated with immune evasion in human triple-negative breast cancer (2020)
Figure 1

Figure 1. Cellular composition of five triple-negative breast carcinomas.

a, Schematic highlighting the application of our single-cell RNA sequencing experimental and analytical workflow for primary patient tissue.

b, UMAP visualisation of 4,986 epithelial cells aligned using canonical correlation analysis in Seurat. Cells are coloured by their cell type annotation (left) and patient of origin (right).

c, Log normalised expression of markers for epithelial (EPCAM), mature luminal epithelial (ESR1), myoepithelial (KRT5, KRT14 and ACTA2) and proliferating cancer cells (MKI67).

d, UMAP visualisation of 19,285 stromal and immune cells aligned and visualised as represented in b.

e, Log normalised expression of markers for fibroblasts (PDGFRB, THY1, COL1A1, ITGB1 and S100A4), endothelial cells (PECAM1), T-cells (CD3D), CD8 T cells (CD8A), T-regulatory cells (FOXP3), B-cells (MS4A1), myeloid cells (CD68) and plasma cells (JCHAIN).

f, Proportion of cell types across each patient.
Stromal cell diversity associated with immune evasion in human triple-negative breast cancer (2020)
Figure 3

Figure 3. Polarised gene regulatory states between cancer-associated fibroblasts and perivascular-like subclasses.

a, Polarised gene regulatory states underlying stromal subclasses. Heatmap shows the averaged regulon activity (area under the curve; AUC) for the top 50 highest TFs regulons as estimated using SCENIC. All regulons are statistically enriched across the four subsets (p < 1x10-5 One-way ANOVA). Heatmap is clustered using Euclidean distance and complete linkage.

b, Candidate transcriptional drivers of each CAF and PVL subset. Violin plots showing the log normalised gene expression (left) of the TF and its respective AUC regulon activity (right). TFs ZEB1 and FOXP1 enriched in myofibroblast-like CAFs, EGR2 and TCF7L2 enriched in inflammatory-like CAFs, MEF2C enriched in PVL cells and NR2F2 enriched in immature-PVL cells.
Stromal cell diversity associated with immune evasion in human triple-negative breast cancer (2020)
Figure 4

Figure 4. Morphological, phenotypic and spatial differences underlying stromal heterogeneity.

a, Summary of the markers distinguishing each of the four stromal subpopulations identified in this study.

b, FACS validation in matched patient tissue. Stromal cells are negatively gated for EPCAM (epithelial), CD45 (immune) and CD31 (endothelium) and positively selected for PDGFRβ. Subsequent markers PDGFRα and CD146 (MCAM) are used to distinguish CAFs and PVL cells, respectively. Expression of FAPHIGH, FAPLOW, CD36+ and CD36- are further used to define myofibroblast-like CAFs, inflammatory-like CAFs, immature-PVL cells and differentiated-PVL cells, respectively.

c-d, Immunofluorescence of cultured human CAFs (c) and PVL cells (d) from passage 8, staining for CD34 (CAFs), ⍺-SMA (myCAFs and PVL cells), CD146 (PVL cells) and CD36 (imPVL cells).

e-f, Quantitative analysis of collagen abundance (e) and orientation (f) using second harmonic generation (SHG) from cellular derived matrices from stromal subsets and representative images multiphoton SHG images (n = 3 biological replicates). All SHG intensity values within each replicate were normalised to the SHG intensity of the myCAFs. Error bars represent standard deviation. Statistical significance for collagen abundance (e) was determined using unpaired two-tailed Student's t test with equal standard deviation with p‐values denoted by asterisks: *p < 0.05, **p < 0.01 and ***p < 0.001. After normalization of the orientation peak distributions (f), statistical significant was determined using a Kruskal-Wallis test with Dunn's post-hoc multiple comparisons test (p value < 0.0001).

g-h, Immunohistochemical staining of PDGFRβ, ⍺-SMA, CD34 and CD146 in serial sections cut 4 μm apart from matched cases; Patient-2 (g) and Patient-4 (h). Images were aligned using FIJI. Tumour (T) regions are annotated by the solid yellow line. Co-localisation of CD34 and CD146 was used to distinguish blood vessels (BV), where their differential staining was used to identify CAFs and PVL cells. g, MyCAFs were found to be localised at the invasive stromal interface (insert A), whilst iCAFs were located at distal regions (insert B) with a high abundance of tumour infiltrating lymphocytes (TILs). h, Case with a high abundance of perivascular-like (PVL) cells in regions surrounded by blood vessels.

i, Validation of disseminated PVL cells from blood vessels using co-immunofluorescence of CD31 (red), CD146 (green) and DAPI (blue). Representative images from Patient-4 is shown.
The Disulfide Catalyst QSOX1 Maintains the Colon Mucosal Barrier
by Regulating Golgi Glycosyltransferases (2022)
Figure 1

Figure 1. QSOX1 KO mice are hypersensitive to induced colitis and show alterations in their microbiome.

Immunofluorescence labeling of QSOX1 and a Golgi marker (GM130) in epithelial cells isolated from murine colon. QSOX1 is green, GM130 is red, and DAPI staining of nuclei is blue in the merged image. Scale bar is 10 µm.

Top, average colon length of WT and QSOX1 KO mice following indicated treatments. For untreated mice, colon lengths were calculated for 3 WT and 3 KO. For treated mice, lengths were calculated from 5 mice except for the DSS-treated WT, for which 3 mice were analyzed. Error bars are standard deviation. Statistical analysis was performed using Tukey multiple comparison of means (** p<0.01). Administration of QSOX1 inhibitory antibody MAb316.1 (αQSOX1), (Grossman et al., 2016; Feldman et al., 2020) did not enhance the sensitivity of WT colons to DSS (p>0.4 for MAb316.1-treated WT vs. WT control antibody (IgG)-treated; p>0.5 for MAb316.1-treated WT vs. WT without antibody treatment). Bottom, representative images of colons following DSS treatment. Scale bar is 1 cm.

Representative colon cross-sections stained with H&E show severe damage to the epithelium in DSS-treated KO mice. Scale bar is 500 μm.

Damage to epithelium and immune cell infiltration in representative DSS-treated QSOX1 KO colons. Scale bar is 50 μm.

Principal coordinate plot of weighted Unifrac data from amplicon sequencing of feces from 5 WT and 5 KO mice using 16S universal eubacterial primers. The three vectors presented exhibit more than 70% of the variation among the groups. ANOSIM R statistic is 0.3, p-value 0.011.

Mean percentage is displayed for genera that differ significantly between WT and KO fecal samples and that represent at least 1% of the sequences for either genotype. This experiment was done once, with 6 mice per group.

Fluorescence in situ hybridization of 16S rRNA (red). Blue indicates DAPI staining of nuclei. Scale bar is 10 µm. Representative images are shown from labeling of 5 WT and 5 KO mice. The experiment was performed twice on different sections from the same set of mice.
The Disulfide Catalyst QSOX1 Maintains the Colon Mucosal Barrier
by Regulating Golgi Glycosyltransferases (2022)
Figure 3

Figure 3. Normal mucin and Vwf polymerization in QSOX1 KO mice.

Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well-packed goblet cell granules in WT and QSOX1 KO colons. Scale bar is 5 µm.

Alcian blue and PAS staining of reduced guanidine-insoluble mucins from WT and QSOX1 KO mice, separated on a 6% polyacrylamide gel.

Amino acid sequences of murine Muc2 (Uniprot Q80Z19), Muc5b (Uniprot E9Q5I3), and Vwf (Uniprot Q8CIZ8) showing sequence conservation in the regions of the cysteines participating in intermolecular disulfide bonding to form polymers. Cysteines are highlighted in yellow, and asterisks indicate those that make intermolecular disulfides. Numbers indicate the amino acid positions of intermolecular disulfide-bonding cysteines in the amino-terminal region of murine Muc2, and in parentheses are corresponding amino acid positions in human MUC2, for reference to panel G.

Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels.

Western blot analysis of blood Vwf from WT and QSOX1 KO mice separated on agarose gels.

QSOX1 immunofluorescence (green) in control (siControl) and QSOX1 knockdown (siQSOX1) MDA-MB-231 cells. Blue is DAPI staining of nuclei. Scale bar is 20 µm.

Western blot analysis of the MUC2 N-terminal region and indicated cysteine mutants in supernatants of transfected cell cultures. No differences in disulfide-mediated dimerization were observed for any MUC2 variant between siC (siControl) and siQ (siQSOX1).

Data information: In panels B, D, and E, each lane corresponds to a sample from one mouse. The results described in panels F and G are representative of 3 experiments that were performed.
The Disulfide Catalyst QSOX1 Maintains the Colon Mucosal Barrier
by Regulating Golgi Glycosyltransferases (2022)
Figure 4

Figure 4. QSOX1 oxidizes Golgi glycosyltransferases.

Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice. Lysates were treated with PEG-mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1.

As for panel A but using antibodies to St3gal1.

As for panel A but using antibodies to B3galt5.

Structure of the human ST6GAL1 catalytic domain in complex with CMP (PDB ID: 4JS2) (Kuhn et al., 2013) with cysteine side chains shown as spheres and numbered. Numbering according to the murine St6gal1 sequence is indicated in parentheses.

QSOX1 oxidizes St6gal1 in vitro. MAb492.1 is a monoclonal antibody that inhibits human QSOX1 (Grossman et al., 2013). St6gal1 with one pair of free cysteines is modified by two PEG-mal additions (2PEG), whereas protein with two pairs of free cysteines acquires four PEG-mal additions (4PEG). The change in migration per two PEG-mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs. protein (Javitt et al., 2020b).

QSOX1 oxidation of St6gal1 mutants. The C350-C361 disulfide (the redox-active disulfide present in the C139A-C403A mutant) is oxidized rapidly, while the C142-C406 disulfide (present in the C350A-C361A mutant) is oxidized slowly.

Data information: For the experiments in panels A-C, equal amounts of total protein were applied to each gel lane, and no reducing agent was added. Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.