EMBO/soda-nel huggingface dataset:
STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation (2019)
Figure 5

Figure 5. Reciprocal role of STAT4 and STAT3 on gene regulation in immune cells.

A Heatmap showing Th17 signature genes (left) or Th17 regulatory genes (right) up- or downregulated in WT or Stat4-KO LPLs by LIF treatment (n=2).

B, C Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice (n=3 per group).

D Quantitative mRNA expression analysis of the indicated genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF.

E, F The GAS-luciferase reporter activity assay was performed in Stat3-/- MEFs transfected with empty vector (EV) or STAT4 constructs and then treated with LIF for 8 hours.

G, H Relative SIE-(I) or AGG-luciferase reporter (J) activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours. The data are representative of three independent experiments (D-H).

I The Il17a/Il17f locus, including qPCR primer sites covering SIEs (purple triangles) or AGG elements (green triangles).

J ChIP analysis of naïve CD4+ T cells obtained from IL-17A-eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. GFP+ cells isolated by flow cytometry were restimulated with IL-6 in the presence or absence of LIF, crosslinked with formaldehyde and immunoprecipitated with anti-STAT3 (top) or anti-STAT4 (bottom) antibodies, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.

K ChIP analysis of naïve WT or Stat4-KO CD4+ T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. The cells were restimulated with IL-6 or LIF for 30 minutes, crosslinked with formaldehyde and immunoprecipitated with the anti-STAT3 antibody, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA. The data are representative of two independent experiments (J-K)

L Relative Il17a-SIE- (left) or AGG-luciferase reporter (right) activities in Stat3-/- MEFs transfected with EV, STAT3 or STAT4 or cotransfected with STAT3 and STAT4 and then treated with IL-6 or LIF for 8 hours.

M Relative Il17a promoter-luciferase reporter activities in Stat3-/- MEFs transfected with the indicated constructs and then treated with LIF for 8 hours. The data are representative of three experiments (L-M). *p< 0.05, **p< 0.01 and ***p< 0.001 (Student's t-test). Error bars represent the SEM.
STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation (2019)
Figure 6

Figure 6. STAT3 is responsible for YAP gene expression and cell proliferation in the intestinal.

A-C Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A. The number of proliferating cells per crypt in (A) (upper panel) or apoptotic cells per field in (B) was determined (n=5 per group, 5 crypts were counted per mouse). Scale bar, 50μm.

D, E Quantitative mRNA expression analysis of the indicated genes in colon tissues obtained from WT or Stat4-KO colitis mice treated as described in Figure 2A (n=3 per group).

F Immunoblot analysis of the expression and modification of the indicated proteins in DLD-1 cells treated with LIF (20 ng/ml) for different durations.

G Immunoblot analysis of YAP expression in WT or Stat4-KO IECs treated with LIF (20 ng/ml) for different durations; β-actin was used as a loading control (top). The statistical analysis of YAP protein intensity is shown in the bottom panel. The data are representative of three experiments.

H The human and mouse Yap promoter locus contains multiple AGG elements, as indicated.

I Relative YAP promoter-luciferase reporter activities in HEK293T cells transfected with EV, STAT3 or STAT4 and then treated with IL-6 or LIF for 8 hours. The data are representative of three experiments.

J, K RNA sequencing analysis of the gene expression profile in IECs treated with or without LIF (50 ng/ml) for 6 hours. Statistical analysis of the average fragments per kilobase of transcript per million mapped reads (J). Heatmap showing the expression profile of cell proliferation-related genes in LIF-treated or control IECs (Ctrl) (K). *p< 0.05, **p< 0.01 and ***p< 0.001 (Student's t-test). Error bars represent the SEM.
STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation (2019)
Figure 7

Figure 7. LIF treatment modulates the outgrowth of the proinflammatory microbiota during intestinal inflammation.

A Principal coordinate analysis (PCoA) of the microbiota composition determined by 16S rRNA gene sequencing of day 0 and day 9 fecal specimens from mice treated as described in Figure 5A (n=5 or 6 per group). Each symbol represents an individual mouse. PC1 and PC2 represent principal components 1 and 2, respectively.

B The bars depict the average relative abundance of the bacterial phyla of the microbiota in fecal specimens, as determined by taxon-based analyses as described in (A) (n=5 per group).

C 16S rRNA sequencing analyses of the Proteobacteria distribution in feces from WT or Stat4-KO colitis mice. (n=5 per group).

D Heatmap depicting the relative abundance of microbes at the family level (top) and the genus level (bottom) in the fecal specimens described in (A) (n=3 per group).

E qPCR of the 16S rRNA genes of microbes in the phylum Proteobacteria, family Enterobacteriaceae, and genera Escherichia-Shigella in the feces of the indicated mice on day0 and day10 (n=6 per group) of colitis induction.

F Quantitative mRNA expression analysis of proinflammatory genes in the colon of mice (n=3 per group) treated as described in Figure 2A. **p< 0.01 and ***p< 0.001 (Student's t-test). Error bars represent the SEM.
Loss of PIKfyve drives the spongiform degeneration in prion diseases. (2021)
Figure 1

Figure 1: PIKfyve is universally depleted in prion diseases.

A: Conspicuous downregulation of PIKfyve, but not FIG4 or VAC14, in brain lysates of terminally sick mice intracerebrally inoculated with prions (RML). Control: mice inoculated with non-infectious brain homogenate (NBH). Right: Quantification of immunoblot. Each dot represents one mouse. ***: p<0.001; unpaired t-test. Error bars represent s.e.m

B: PIKfyve is selectively downregulated in mice infected with ME7 prions. Right: Quantification. Each dot represents one individual (n=4/group). ***: p<0.001; unpaired t-test. Error bars represent s.e.m.

C: Time course of PIKfyve downregulation in brains of prion-infected mice (3 mice/time point). PIKfyve (but not FIG4, calnexin, NeuN and synaptophysin) was suppressed between 60 and 120 dpi. T: terminal disease stage.

D: The PIKfyve/FIG4 ratio gradually decreased in prion-infected mice (unpaired t-test). **: p<0.01; ***: p<0.001. Each dot represents one mouse.

E: Prion-infected Gt1 cells (75 dpi) stained with Alexa-Fluor647-succinimidyl ester which stains vacuoles negatively (arrows).

F: Loss of PIKfyve, but not FIG4 and VAC14, in prion-infected Gt1 cells. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; unpaired t-test. Error bars represent s.e.m.

G: tga20 COCS were treated with pooled IgG or POM1 (72 hours). POM1 suppressed PIKfyve but not FIG4 and VAC14. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; ***: p<0.001; unpaired t-test Error bars represent s.e.m.

H: Immunoblots of human CJD brains (red: sampled region). Gt1Sc: prion-infected Gt1 cells. Loading controls: actin and calnexin (Cnx)

I: Quantification of immunoblots revealed moderate and substantial PIKfyve loss in Type-1 and Type-2 CJD, respectively (unpaired t-test). **: p<0.01; ***: p<0.001. Error bars represent s.e.m.

J: Brain homogenates from panel C were immunoprecipitated with anti-VAC14 antibodies. FIG4 and PIKfyve did not co-precipitate in prion-infected brains at terminal disease.
Loss of PIKfyve drives the spongiform degeneration in prion diseases. (2021)
Figure 2

Figure 2: ER stress regulates PIKfyve expression levels.

A : Time course of eIF2α and PERK phosphorylation in prion-inoculated C57BL/6 mice. T: terminal disease.

B: PIKfyve/eIF2α and eIF2α(P)/eIF2α ratios assessed from Western blots as in A (n=3/group/time point). PIKfyve levels gradually decreased after 60 dpi whereas eIF2α became phosphorylated from 30 dpi until terminal disease. eIF2α phosphorylation and pPIKfyve depletion in POM1-treated tga20 COCS. **: p<0.01; ***: p<0.001; unpaired t-test; Error bars represent s.e.m.

C: Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment. Each dot represents an individual experiment. Statistics: Unpaired t-test. **: p<0.01.

D: Transient PIKfyve suppression in thapsigargin-treated Gt1 cells with recovery after 6 hours. PERK phosphorylation confirmed ER stress.

E: Gt1 cells were inoculated with NBH or prions (60 dpi), pulsed with [35S]Met/Cys (20 min, 37 °C) and immediately lysed or chased without 35S (37 °C, ≤3 hours). For control, cells were treated with scrambled siRNA or siRNA against PIKfyve. Proteins were immunoprecipitated with antibodies to PIKfyve and subjected to SDS-PAGE and autoradiography (n=3 individual biological replicates).

F: Autoradiographic signals expressed as percentage of the initial PIKfyve levels. Prion infection shortened the half-life of PIKfyve from 144 to 52 min. First-order kinetics was assumed. Error bars represent s.e.m and were calculated based on three individual biological replicates.

G: Left: partially restored PIKfyve levels in prion-infected tga20 mice treated with LIN5044 (LCP) or vehicle. Right: quantification of the western blot. Each dot represents an individual biological replicate. *: p0.05; unpaired t-test Error bars represent s.e.m.

H: Left, tga20 COCS were inoculated with RML or NBH, optionally treated with GSK2606414 starting at 21 dpi, and lysed at 35 dpi. Depletion of PIKfyve in prion-infected samples was largely rescued by GSK2606414 (GSK). Right, quantification of western blot. Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01; unpaired t-test; Error bars represent s.e.m.
Loss of PIKfyve drives the spongiform degeneration in prion diseases. (2021)
Figure 3

Figure 3: Prion infection causes deacylation and degradation of PIKfyve.

A: Acyl-rac captured hydroxylamine-cleavable, acylate PIKfyve from mouse brains. Control: TRAPα, a non-acylated protein. Input: 10% of lysate.

B: Brain lysates from prion-inoculated mice (60 dpi) precipitated by Acyl-rac. PIKfyve, but not calnexin (Cnx), was deacylated in prion-infected samples. Each lane represents an individual mouse. Lower panel: lysate used for Acyl-rac.

C: Autoradiography of Gt1 cells transfected with PIKfyve-GFP or PIKfyveΔacyl (24h), metabolically labeled with 3H-palmitate (2 hours), and immunoprecipitated with anti-GFP antibodies. Lower panel: Western blot on immunoprecipitates using anti-GFP antibodies. Error bars represent s.e.m. (n=3 individual biological replicates).

D: Gt1 cells transiently expressing PIKfyve-GFP or PIKfyveΔAcyl (24h) were subjected to a [35S]Met/Cys pulse (20 min, 37 °C) and immediately lysed or chased (60 min). Proteins were immunoprecipitated with anti-GFP antibody and subjected to SDS-PAGE and autoradiography. Lysates from cells treated with siRNA against PIKfyve were also pulsed and immunoprecipitated and subjected to autoradiography to ensure correct band is detected. Error bars represent s.e.m. (n=3 individual biological replicates).

E, F: Acyl-rac of Gt1 cells subjected to siRNA against zDHHC9, zDHHC21 or both (72 hours post transfection), showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 (quantified in F). Each dot represents a separate experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m.

G: Gt1 cells were transfected with siRNA against zDHHC9+21 and stained with LAMP1. Arrows: vacuoles.

H-I: Flag-tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding. After 72 hours, zDHHC9/21 (magenta) were localized to the Golgi apparatus (GM130+, cyan) in NBH-exposed cells but not in prion-infected cells. n=3 individual biological replicates.
Loss of PIKfyve drives the spongiform degeneration in prion diseases. (2021)
Figure 4

Figure 4: Loss of PIKfyve induces lysosomal defects.

A: Upper row: prion-infected Gt1 cells (75 dpi) immunostained for LAMP1 showing prominent vacuoles. Control: NBH-inoculated cells. Middle row: Gt1 cells transfected with shRNA targeting PIKfyve or luciferase (control) for 5 days and immunostained for LAMP1. Depletion of PIKfyve resulted in LAMP1+ vacuoles (yellow). DAPI: nuclear stain. Lower row: prion-infected and NBH treated cells (75 dpi) contained LAMP2- vacuoles. n=3 individual biological replicates.

B: Prion-infected tga20 COCS (45 dpi) immunostained with NeuN and LAMP1. Control: NBH-exposed COCS. Nuclei: DAPI. Prion infection induced LAMP1 upregulation in neurons (quantification: Appendix Fig. S4A) (n=3 individual biological replicates).

C: Higher magnification showing LAMP1+ vacuoles (arrowheads) in prion-infected COCS.

D: tga20 COCS treated with POM1 or control IgG (10 days) and immunostained with NeuN and LAMP1. Nuclei: DAPI. POM1-treated COCS showed increased LAMP1 expression (quantification: Appendix Fig. S4B). NeuN+ cells exhibited vacuoles (arrowheads).

E: Gt1 cells were transfected with control siRNA (scrambled) or siRNA targeting PIKfyve for up to 72 hours and stained with LysoSensor Yellow/Blue. Cells were gated based on emission spectra, and mean fluorescence intensities (MFIs) per sample were quantified. Increased 530/450 MFI ratio indicates enrichment of acidic compartments. No acidic compartment expansion in PIKfyve-depleted cells. n=4 individual biological replicates. Error bars represent s.e.m.

F: β-hexosaminidase A activity in brain lysates from terminally scrapie-sick and NBH-inoculated mice. Control: NBH inoculated mice. Prion-infected brain lysates showed 2-2.5 fold increased activity. Panels show independent triplicates. Statistics: unpaired t test. **: p<0.01. Error bars represent s.e.m.

G: Lysosomal enzymes, but not β-actin, were elevated in brains of terminally scrapie-sick mice. Panels show independent triplicates. Statistics: ANOVA.. **: p<0.01. Error bars represent s.e.m.
Loss of PIKfyve drives the spongiform degeneration in prion diseases. (2021)
Figure 5

Figure 5: Prions induce lysosomal dysregulation via TFEB.

A: Time course of TFEB phosphorylation in prion-infected C57BL/6 mice. Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥90 days. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. *: p0.05; **: p<0.01. Error bars represent s.e.m.

B: TFEB-controlled transcripts in brains of terminally scrapie-sick C57BL/6 mice were measured by qPCR. Prion infection led to upregulation of these genes, but not of STAT3. Control: NBH-inoculated mice. ANOVA on independent triplicates. ***: p<0.001. Error bars represent s.e.m.

C: Prion-infected Gt1 cells (75dpi) showed reduced TFEB phosphorylation. Total levels of TFEB remain unchanged. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m.

D: COCS prepared from tga20 mice were infected with RML prions and cultured for 5 weeks. Cell lysates were prepared at 4 wpi followed by Western blot analysis using anti-phospho-TFEB antibody. Activated (dephosphorylated) TFEB was increased in RML infected COCS. Total levels of TFEB remain unchanged. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. Error bars represent s.e.m.

E: Gt1 cells (as in D) were fixed and stained with anti-TFEB antibody. Prion-infected cells showed nuclear translocation of TFEB. DAPI: Blue. Quantification: Each dot represents an individual biological replicate (30 images were quantified per replicate). Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m.

F: TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions (75 dpi). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m.

G: TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl (shRNA against luciferase). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m.
Loss of PIKfyve drives the spongiform degeneration in prion diseases. (2021)
Figure 6

Figure 6: PI(3,5)P2 attenuates spongiosis and lysosomal dysfunction.

A: Transient overexpression of Flag-tagged zDHHC9+21 (72 hours) restored PIKfyve levels in prion-infected Gt1 cells. Right: Western blot quantification. Each dot represents an individual experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m.

B: Cotransfection of zDHHC9+21 reduced vacuolation in prion-infected Gt1 cells. Each dot represents a separate experiment (1000 cells/experiment, χ2: p<0.001). **: p<0.01; unpaired t-test. Error bars represent s.e.m.

C: Prion-infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi. Control: empty lentiviral vector. At 79 dpi, GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels. Right: Quantification of western blots (n=3; unpaired t-test). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.

D: Number of vacuolated cells from the experiment shown in C. GADD34 expression rescued the total number of vacuolated cells (1000 cells/experiment, χ2: p<0.001). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.

E: tga20 COCS were infected with RML and optionally treated with bPIP (5µg/ml). At 45 dpi, NeuN morphometry revealed ablation of cerebellar granule layer (CGN) in prion-infected slices and its rescue by bPIP. Control: NBH treated COCS. Each dot represents an individual slice (Statistics: ANOVA). *: p<0.05. **** p<0.0001.

F: tga20 COCS were treated with POM1 (optionally pre-blocked with recPrP) and treated with bPIP (5µg/ml). At 14 dpi NeuN morphometry revealed POM1-induced ablation of cerebellar granule layer (CGL) and rescue by bPIP.

G: Prion-infected Gt1 cells were treated with bPIP for 3 days. The number of vacuolated cells was reduced (1000 cells/experiment, χ2: p<0.001); Statistics: Chi-square test. Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.
Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis (2020)
Figure 1

Figure 1: Acute inhibition of ribosome synthesis and function in vivo reduces translation of components of the translational machinery and decreases the abundance of polysome-associated, energy metabolism-related mRNAs

(A) Western analysis for on-target effects for Everolimus (EV, P-RPS6) and CX-5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX-5461 vehicle: 25 mM NaH2PO­­­4) (V/V; mouse #1-6), 5 mg/kg everolimus (EV; mouse #7-12), 35 mg/kg CX-5461 (mouse #13-18) or both drugs (CX-5461+EV; mouse #19-24) for 2 hours (n=6 per treatment group). Actin was used as a loading control.

(B) A schematic illustration of the polysome profiling analysis workflow: cytoplasmic lysate was layered on top of a linear 10-40% sucrose gradient, ultracentrifuged (36,000 rpm, 2¼ hours, 40C) and fractionated using the Foxy Jr Fraction Collector with constant monitoring of absorbance at 260 (A260) nm by an ISCO UA-6 Absorbance Detector. Fractions (one fraction per minute: 800 µL per tube) corresponding to polysomal mRNAs that were bound by four or more ribosomes were pooled and analyzed by RNA-seq followed by data analysis using anota2seq or limma.

(C) Enrichment analysis by MetaCore® GeneGO of genes in "translation up" (red) and "translation down" (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX-5461+EV treatment group with the V/V group (n=6). "Ratio" values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore®'s database.

(D) Genes implicated in "Translation: initiation" and "Translation: Elongation-Termination" processes based on Fig 1C and Fig EV1E. log2FC: log2 fold change; FDR: false discovery rate (adjusted P value).

(E) Activity levels of key biological processes involved in cellular growth, proliferation and metabolism based on single sample gene set enrichment analysis (ssGSEA) of indicated comparisons. Percentage increase (red) or decrease (blue) in the activity levels of key biological processes involved in cellular growth, proliferation and metabolism based on ssGSEA of indicated comparisons. Data were obtained from n=6 mice per treatment group.
Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis (2020)
Figure 3

Figure 3: cAMP-dependent pathway mediates resistance to CX-5461-everolimus co-treatment

(A) Enrichment analysis by GeneGO MetaCore® of the polysomal RNAseq data comparing CX-5461+EV-resistant (CMB) and drug-naïve cells (CTRL) (n=3; false discovery rate (FDR) ≤ 0.05; fold change (FC) ≥ 1.5 or ≤-1.5).

(B) Abundance of intracellular 3'5'-cyclic AMP as measured by a liquid chromatography (LC)-mass spectrometry (MS) analysis. Graphs represent mean ± SEM of n=3 (5-6 technical replicates each). Data were analyzed by one-way ANOVA. CTRL vs EV, p = 0.8951; CTRL vs CX, p = 0.0871; CTRL vs CMB, p = 0.0012.

(C) Western analysis of EPAC1 and EPAC2 abundance, as well as active GTP-bound RAP1 levels in the indicated early-passage cells during their log-phase growth period (n=3). Actin and total RAP1 were used as loading controls. Quantitations of the blots are shown in Appendix Fig S3A-C.

(D) Propidium iodide (PI) exclusion assays of CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours.

(E) PI exclusion analysis of early-passage drug-naïve (CTRL) lymphoma cells treated with CX-5461 and everolimus in the presence or absence of selective PKA activator 6-Bnz-cAMP for 48 hours.

(F) PI exclusion analysis of the CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI-05 for 48 hours.

(G) PI exclusion analysis of early-passage drug-naïve (CTRL) cells treated with CX-5461 and everolimus in the presence or absence of the selective EPAC activator 8-pCPT-2-O-Me-cAMP for 48 hours.

Data information: (D, E, F, G) Data (n=3) were analyzed by paired one-way ANOVA. Green triangle: CX-5461-everolimus combination therapy-resistant (CMB) cells clone #8; green square: CMB cells clone #9; green circle: CMB cells clone #31. Blue circle: early-passage drug-naive (CTRL) cells clone #6; blue square: CTRL cells clone #33; blue triangle: CTRL cells clone #38. ns, not significant, P≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01.