EMBO/soda-nel huggingface dataset:
Multiple cancer pathways regulate telomere protection (2019)
Figure 5

Figure 5. Phosphorylation of TRF1 residue T330 stabilizes TRF1 telomeric foci in vivo. (A) Trf1lox/lox p53-/- MEFs were transduced with eGFP-tagged Trf1 WT or mutant alleles to overexpressed TRF1 depicted variants. Endogenous TRF1 was deleted by transduction with Cre recombinase. Opera high-content screening (HCS) system was used to quantify the GFP spot intensity per cell. (B) Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. (C) Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=15 biological replicates (D) Growth curves of Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicates (E) Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. (F) Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicates (G) Western blot images of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed treatment with ERKi. Data are representative of n=2 biological replicates (H) Representative images (above) and percentage (bottom) of Telomeric and 53BP1 colocalizing foci (TIFs) per cells of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT and the indicated mutants upon treatment with the ERKi. White arrowheads: colocalization of telomeric and 53BP1. Scale bars, 10μm. Data are representative of n=2 independent experiments. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.
Multiple cancer pathways regulate telomere protection (2019)
Figure 6

Figure 6. Genetic model of the telomeric role of ERK1/2-mediated TRF1 phosphorylation (A) Western blot image (above) and quantification (bottom) of TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. Data are representative of n=3 independent experiments. (B) Representative images (above) and quantification (bottom) of TRF1 telomeric foci in ERK1/2 RNA interfered p53-/- MEFs. Scale bars, 5μm. Data are representative of n=2 independent experiments. (C) p53-/- MEFs were sequentially transduced with lentiparticles encoding short hairpins against ERK1/2 and retroparticles for eGFP-tagged Trf1 WT or mutant alleles to overexpress TRF1 depicted variants in the absence of ERK1/2. (D) Western blot image of eGFP-tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. (E) Representative images (above) and percentage (bottom) of telomeric and 53BP1 colocalizing foci (TIFs) per cell in p53-/- MEF with or without overexpression of eGFP-Trf1 WT and indicated mutants upon genetic depletion of ERK1/2. White arrowheads: colocalization of telomeric and 53BP1.Scale bars, 10μm. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01.
Multiple cancer pathways regulate telomere protection (2019)
Figure 7

Figure 7. In vitro combinatorial studies of PI3Ki with novel TRF1 inhibitory compounds. (A-F) Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates. (G-L) Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of n=3 (Combination in K), n=4 (Combination in G, L), n=5 (RTKi in G, Combination in H, I, J, Docetaxel in L), n=6 (DMSO in G, H, I, J, K, L and ERKi in H), n=11 (PI3Ki in G, H, I, J, L) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.
Multiple cancer pathways regulate telomere protection (2019)
Figure 8

Figure 8. In vivo combinatorial studies of PI3Ki with novel TRF1 inhibitory compounds in patient-derived GBM xenograft models. (A-D) Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in A: vehicle n=16, PI3Ki n=8, ERKi n=16, Combination n=8; in B: Vehicle n=16, PI3Ki n=16, MEKi n=8, Combination n=8; in C: Vehicle n=8, PI3Ki n=16, Docetaxel n=16, Combination n=8; in D: Vehicle n=16, PI3K n=16, Gemcitabine n=4, Combination n=4. Pvalues represent the mean of all the time points (E-H) Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors: in E: vehicle n=15, PI3Ki n=10, ERKi n=8, Combination n=2; in F: Vehicle n=15, PI3Ki n=10, MEKi n=8, Combination n=6; in G: Vehicle n=15, PI3Ki n=10, Docetaxel n=8, Combination n=4; in H: Vehicle n=15, PI3K n=10, Gemcitabine n=8, Combination n=6. Significant differences using unpaired t‐test with Welch`s correction are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.
Suppression of the HSF1-mediated proteotoxic stress response by the metabolic stress sensor AMPK. (2015)
Figure 1

Metformin suppresses the HSF1-mediated PSR

A The connectivity map suggests that metformin generates a gene expression pattern concordant with HSF1 inactivation. Perturbagens inducing and reversing the biological state encoded in the query signature are assigned positive and negative enrichment scores, respectively.

B Following treatment with 10 mM metformin overnight, NIH3T3 reporter cells were heat-shocked at 43°C for 30 min and recovered at 37°C for 5 h. GFP induction was quantitated by flow cytometry. HS: heat shock.

C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting.

D, E Following heat shock and recovery for 4 h, mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT–PCR (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). β-actin was used as the internal control.

F Reporter cells were treated with 10 mM metformin overnight, and viability was measured using Guava ViaCount® reagents (mean ± SD, n = 5, n.s., not significant, Student's t-test).

G Left panel, graphic depiction of the ELISA-based DNA binding assay. HSE: heat shock element. Right panel: Immediately following heat shock at 43°C for 30 min, nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). Experimental details are described in Materials and Methods.

H Graphic depiction of the adapted PLA. A pair of primary antibodies recognizes DNA-bound HSF1s and dsDNAs, respectively.

I HEK293T cells stably expressing a scramble or HSF1-targeting shRNA were treated with 10 mM metformin overnight and heat-shocked at 43°C for 30 min. Cells were fixed for PLA to detect HSF1–DNA interactions. Experimental details are described in Materials and Methods. Scale bars: 50 μm for low magnification and 10 μm for high magnification.

J Immediately following heat shock, cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting. LDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. C: cytoplasm; N: nucleus.

K Reporter mice (ventral side shown) were pre-treated with metformin for 3 days, 2 mg/mouse/day, via i.p. injection. Following a single i.p. injection of velcade (5 mg/kg) and recovery for 6 h, whole-body luciferase activities were quantitated by bioluminescence imaging. The photon flux of each mouse was expressed as photon/s/cm2/steradian (right panel, mean ± SD, n = 3, one-way ANOVA, *P < 0.05; **P < 0.01).

Source data are available online for this figure.
Suppression of the HSF1-mediated proteotoxic stress response by the metabolic stress sensor AMPK. (2015)
Figure 2

AMPK suppresses HSF1 activation through physical interaction

A-C Reporter cells were treated with and without 1 μM A-769662 for 3 h followed by heat shock at 43°C for 30 min and recovery at 37°C for 5 h. Hspa1a and Hspb1 mRNA levels were quantitated by qRT–PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001). ACC phosphorylation was detected by immunoblotting (C).

D-F Primary MEFs were derived from Ampkα1fl/fl; Ampkα2fl/fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1/2 in vitro. AMPKα levels were detected 4 days after transductions (D). Following viral transductions, Hsp mRNA levels were quantitated by qRT–PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43°C heat shock for 30 min and recovery for 4 h (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).

G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids: the HSF1-dependent reporter pHSE-SEAP, in which ideal HSEs drive the expression of secreted alkaline phosphatase (SEAP), and the control reporter pCMV-Gaussia Luc, in which a CMV promoter drives the expression of secreted Gaussia luciferase. In HEK293T cells, either LacZ or GST-AMPKα1CA plasmid was co-transfected with the two reporter plasmids. After 24 h, the transfected cells were heat-shocked at 44°C for 45 min. The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock. SEAP signals were normalized to Gaussia luciferase signals (mean ± SD, n = 5, Student's t-test, **P < 0.01, ***P < 0.001).

H, I Following treatment with 10 mM metformin overnight, endogenous AMPKα and HSF1 proteins were co-precipitated using anti-AMPKα agarose conjugates from lysates of immortalized Hsf1+/+ MEFs (H). HC: heavy chain. Endogenous HSF1-AMPKα interactions in immortalized Hsf1+/+ and Hsf1−/− MEFs were visualized in situ by PLA (I). Experimental details are described in Materials and Methods. Scale bars: 50 μm.

Source data are available online for this figure.
Suppression of the HSF1-mediated proteotoxic stress response by the metabolic stress sensor AMPK. (2015)
Figure 3

AMPK phosphorylates Ser121 to inactivate HSF1

A In HEK293T cells that stably express an shRNA targeting the 3′ UTR of human HSF1, HSF1WT or HSF1S121A was expressed. Following treatment with 10 mM metformin overnight, the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho-HSF1 Ser121 antibody (A8041, Assay Biotechnology).

B Primary Ampkα1fl/fl; Ampkα2fl/fl MEFs were transduced with adenoviral GFP or Cre. Following treatment with 10 mM metformin overnight or treatment with 10 μM A-769662 for 3 h, HSF1 Ser121 phosphorylation was examined by immunoblotting.

C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting.

D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight. Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His-tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C (CC). HSF1 Ser121 phosphorylation was detected by immunoblotting.

E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ or AMPKα1CA (E). Following expression of HSF1WT or HSF1S121A, cells were treated with and without 10 mM metformin overnight (F) (mean ± SD, n = 5, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).

G, H FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with LacZ or AMPKα1CA plasmids into HSF1-deficient HEK293T cells for 3 days (G). Following transfection with FLAG-HSF1WT or FLAG-HSF1S121A plasmids for 3 days, HSF1-deficient HEK293T cells were treated with and without 10 mM metformin overnight (H). Nuclear proteins were extracted to measure HSF1–DNA binding by the ELISA-based DNA binding assay using anti-FLAG antibodies (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).

I, J Following transfections and metformin treatment as described in (G) and (H), cytoplasmic and nuclear proteins were extracted for immunoblotting using anti-FLAG antibodies.

Source data are available online for this figure.
Suppression of the HSF1-mediated proteotoxic stress response by the metabolic stress sensor AMPK. (2015)
Figure 4

Metabolic stress suppresses the PSR through AMPK

A, B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine (A) or 4.5 g/l glucose (B). Following heat shock at 43°C for 30 min and recovery for 5 h, GFP induction was quantitated by flow cytometry.

C, D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA-based DNA binding assay (mean ± SD, n = 3, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001).

E, F Following nutrient deprivations overnight, the reporter cells were heat-shocked at 43°C for 30 min and recovered overnight. Individual proteins were detected by immunoblotting.

G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.

I, J HEK293T cells were sequentially transfected with control or AMPKα1/2-targeting siRNAs and dual HSF1 reporter plasmids. 24 h later, transfected cells were starved overnight before reporter activities were measured (mean ± SD, n = 6, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).

K, L FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with the dual HSF1 reporter plasmids into HSF1-deficient HEK293T cells for 2 days, followed by overnight starvation (mean ± SD, n = 5, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).

Source data are available online for this figure.
Suppression of the HSF1-mediated proteotoxic stress response by the metabolic stress sensor AMPK. (2015)
Figure 5

Heat shock suppresses AMPK and interferes with cellular responses to metabolic stressors

A Reporter cells were heat-shocked at 43°C for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.

B Immediately following heat shock at 43°C for 40 min, AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates.

C Following heat shock at 43°C for 40 min, both detergent-soluble and detergent-insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting.

D, E Immediately following heat shock at 43°C for 40 min, HEK293 cells were subjected to nutrient deprivations for 4 h. AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting.

F-H HSF1WT or HSF1S121A plasmids were transfected into HSF1-deficient HEK293T cells. Transfected cells were heat-shocked at 43°C for 40 min. HSP mRNAs were quantitated by qRT–PCR following overnight recovery. HSF1–DNA binding was measured immediately after heat shock (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001).

I Schematic depiction of the opposite impacts of metabolic and proteotoxic stress on AMPK and HSF1. While metabolic stress activates AMPK to suppress HSF1, proteotoxic stress suppresses AMPK to enhance HSF1 activation. Dashed arrow signifies HSF1 activation independent of AMPK.

Source data are available online for this figure.
Suppression of the HSF1-mediated proteotoxic stress response by the metabolic stress sensor AMPK. (2015)
Figure 6

Metabolic stressors inactivate HSF1 and disrupt proteostasis in tumor cells

A, B Following treatment with 10 μM metformin for 3 days, the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP (mean ± SD, n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001). Normal rabbit IgG served as the negative control (A). Following the same treatment, HSP mRNA levels were quantitated by qRT–PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001) (B).

C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT–PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).

E Following treatment with 10 μM metformin for 7 days, protein levels in WM115 and S462 cells were measured by immunoblotting.

F, G Following treatment with 10 μM metformin for 7 days, levels of polyubiquitinated proteins were detected in both detergent-soluble and detergent-insoluble fractions in WM115 and A2058 cells using a Lys48-specific polyubiquitin antibody.

H A2058 cells were treated with 10 μM metformin alone or co-treated with 2 μM compound C for 7 days. Individual proteins were detected by immunoblotting.

I HEK293T cells stably expressing either scramble or HSF1-targeting shRNAs were treated with 10 μM metformin for 7 days. Individual proteins were detected by immunoblotting.

J In HEK293T cells, a plasmid encoding HA-polyQ79-GFP was co-transfected with the indicated plasmids. Following treatment with and without 10 μM metformin for 5 days, the sizes of aggregates in detergent-insoluble fractions were quantitated by a Multisizer™ 3 Coulter Counter. Experimental details are described in Materials and Methods.

K, L Immortalized and RAS-transformed MEFs were treated with either 10 μM metformin (K) or different concentrations of glucose (L) for 7 days. Individual proteins were detected by immunoblotting.

M Following transfection with LacZ or polyQ79, HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size.

Source data are available online for this figure.
Suppression of the HSF1-mediated proteotoxic stress response by the metabolic stress sensor AMPK. (2015)
Figure 7

Metformin inactivates HSF1 to retard tumor growth

A, B A2058 cells were transduced with lentiviral scramble or HSF1-targeting (hA6 and hA9) shRNAs. (A) Cell numbers were quantitated by Hoechst 33342 DNA staining (mean ± SD, n = 4, two-way ANOVA, ***P < 0.001). (B) Protein levels were measured by immunoblotting.

C A2058 cells stably expressing LacZ or HSF1 were grown in medium containing 4.5, 1.0, or 0.45 g/l glucose. Following treatment with 10 μM metformin, cell proliferation was measured by Hoechst 33342 DNA staining (mean ± SD, n = 5, two-way ANOVA, ***P < 0.001).

D, E 1 × 106 LacZ- or HSF1-expressing A2058 cells were transplanted into female NOD/SCID mice. One day after transplantation, metformin was administered via drinking water at 1 mg/ml. Tumor incidence (log-rank test; D) and volumes (E) were measured (mean ± SEM, two-way ANOVA n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Tumor growth curves were fitted to exponential growth models to calculate tumor doubling time (DT).

F Individual proteins were measured by immunoblotting using lysates of three tumors from each treatment group.

G Protein levels were measured by immunoblotting using lysates of mouse liver tissues.

H A2058 cells stably expressing FLAG-HSF1WT or FLAG-HSF1S121A were treated with 10 μM metformin for 7 days. Protein levels were measured by immunoblotting.

I A2058 cells stably expressing HSF1WT or HSF1S121A were grown in Corning®ultra-low attachment 96-well culture plates with 10 μM metformin and different concentrations of glucose for 7 days, 10,000 cells per well. Viable cells were counted by Guava flow cytometer using ViaCount® reagent (mean ± SEM, n = 8, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).

J Tukey box plots showing the inverse correlation between AMPKα Thr172 phosphorylation and HSP mRNA expression in ccRCC. Patients were stratified on the median value of AMPKα Thr172 phosphorylation reverse-phase protein array (RPPA) scores (Student's t-test).

K Schematic depiction of the suppression of HSF1 and disruption of proteostasis by AMPK in malignant transformation.

Source data are available online for this figure.
An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs (2022)
Figure 3

Figure 3: Purification and characterization of iTCE-derived embryonic-like myogenic progenitors (eMPs).

A-F. Flow cytometry quantification for Pax7 and myogenic progenitor surface markers in cells derived from D13 iTCEs.

G,H. Flow cytometry quantification of Pax7, CD34 marking human muscle progenitors with adipogenic potential, and the neural crest marker CD57 in cells derived from D13 iTCEs.

I. Flow cytometry quantification of Pax7, integrin α9, and CD56 in cells isolated from D13 iTCEs.

J. Representative brightfield images of the adhesion of integrin α9 and CD56 positive flow cytometrically sorted eMPs isolated from D13 iTCEs on vitronectin (Vtn), fibronectin (Fn), laminin (Lmn), and gelatine (Gel) in the presence of ROCK inhibitor.

K. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media (PM) after isolation.

L. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media (DM).

M-O. Quantification of the number of Pax7 (M), MyoD (N) and Pax7/MyoD double-positive (O) cells in hskMPs and eMPs in PM and after switching to DM.

P. Representative immunostaining for the muscle fiber marker myosin heavy chain (MyHC) of eMPs maintained in DM.

Q. Quantification of the fusion index of hskMPs and eMPs in PM and DM.

R-T. Quantification of the total myotube area (R), myotube width (S), and myotube length (T) of hskMPs and eMPs in DM.

U-Z, Representative immunostainings and quantification of eMPs in DM expressing Pax7 (V), MyoD (X), and MyoG (Z), as well as the proliferation marker Ki67.

Data information: K,L,P. Left-hand scale bar = 75 μm, right-hand scale bar = 50 μm. J. Scale bar = 40 μm. U,W,Y. Scale bar = 75 μm. M-O,Q-T,V,X,Z, Data are represented as means ± S.E.M from n=4 (M-O,Q) and n=3 (R-T,V,X,Z) independent experiments. A-I. Representative sorts of n=3 repeats are provided in Appendix Table S6. A-Z. hiPSC donor 1. p-values are *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using ANOVA followed by a Bonferroni post hoc test.