EMBO/soda-nel huggingface dataset:
Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1 (2021)
Figure 2

Figure 2: Total body irradiation leads to glomerulosclerosis and glomerular endothelial cell senescence.

A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 5 for RT+ and RT-, respectively.

B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively.

C: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively.

D: p16 mRNA expression in whole kidney from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. n = 5 for RT+ and RT- mice, respectively.

E: p21/griffonia simplicifolia/nephrin coimmunostaining (left panels) and quantification of endothelial p21-positive cells (right panel) in glomeruli of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively.

F: 53BP1/CD34 coimmunostaining (left panels) and quantification of glomerular endothelial cell 53BP1 foci (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Arrows show 53BP1 foci.

G: Relative mRNA expression of PAI-1 in whole kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. n = 5 for RT+ and RT- mice, respectively.

H: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. n = 4 for RT+ and RT- mice, respectively.

Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.
Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1 (2021)
Figure 3

Figure 3: A time course experiment that reveals progressive appearance of senescence and loss of podocytes with age.

A: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4-, 12-, 18- and 24-month-old mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules.

B: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4- and 12-month-old irradiated (RT+) and non-irradiated (RT-) mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules.

C: Bioluminescence (BLI, upper panel) and quantification (lower panel) kidney BLI area in young or old mice (upper panel). n = 3 mice.

D: Bioluminescence (BLI, upper panel) and quantification (lower panel) kidney BLI area in irradiated (RT+) and non-irradiated (RT-) mice over time. n = 3- 6 mice according to the different time points.

Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test.
Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1 (2021)
Figure 4

Figure 4: Clearance of senescent cells prevents glomerulosclerosis, podocyte loss and PAI-1 overexpression during aging.

A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5.

B: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5.

C: PAI-1 immunostaining (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5.

Data information: Data are means ± SEM. Statistical analysis: t-student test: AP+ versus AP- mice.
Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1 (2021)
Figure 5

Figure 5: Endothelial specific PAI-1 inactivation prevents age-associated glomerular lesions.

A: PAI-1 immunohistochemistry (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in kidneys of PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively.

B: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 12 for PAI-1flox and PAI-1∆endo mice, respectively.

C: TUNEL assay (upper panels) and quantification of TUNEL-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 6 for PAI-1flox and PAI-1∆endo mice, respectively.

D: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively.

E: Serum creatinine measurement in PAI-1flox (n = 6) and PAI-1∆endo (n = 7) mice at 22 months of age.

F: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively.

G: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys (upper panels) from PAI-1flox and PAI-1∆endo mice at 22 months of age and quantification (lower panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively.

H: 53BP1/CD34 coimmunostaining (upper panels) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X630. Scale bar = 10 μm. n=4 and n=6 for PAI-1flox and PAI-1∆endo mice, respectively. Arrows show 53BP1 positive foci.

Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.
Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1 (2021)
Figure 6

Figure 6: PAI-1 secretion drives the endothelial cells-podocyte cross-talk during senescence.

A: time of cell population doubling of primary glomerular endothelial cells from passage 8 (p8) to passage 17 (p17).

B: Senescence-associated β-galactosidase staining of glomerular endothelial cells (GEnC) at early (p9) or late (p17) passage.

C: Relative mRNA expression of senescence markers (p16 and p21) and SASP molecules (PAI-1, IL6, IL8) in GEnC at early (p9) or late (p17) passage. n = 5 independent experiments.

D: Scheme of the experimental protocol used for co-culture experiments with glomerular endothelial cells (GEnC) and podocytes.

E: Immunofluorescence of paxilin (green) and phalloidin (red) in podocytes stimulated with control medium, supernatant (SN) from glomerular endothelial cells from early (p9) or late (p17) passage and treated either with vehicle or Tiplaxtinin, a PAI-1 inhibitor (upper panel). Quantification of focal adhesions (lower panel) was performed by counting the number of paxilin-positive spots in 30 cells from three independent experiments. Original magnification X1000. Scale bar = 20 μm.

F: Annexin-V-FITC staining (upper panel) and quantification by FACS (lower panel) of podocytes stimulated with supernatant (SN) from GEnC at early (p9) or late (p17) passage and treated either with vehicle or Tiplaxtinin. n = 4 independent experiments.

Data information: Data are means ± SEM. Statistical analysis: t-student test for C and D; ANOVA followed by the Tukey-Kramer test for F and G.
Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1 (2021)
Figure 7

Figure 7: Glomerular PAI-1 expression correlates with glomerular senescence and predicts outcomes of kidney transplant from elderly donors.

A: PAI-1 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). Original magnification X400. Scale bar = 20 μm.

B: p16 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). The arrows point to the staining. Original magnification X400. Scale bar = 20 μm.

C: PAI-1/CD34 coimmunostaining in old- positive or negative glomerular PAI-1 donors. Original magnification X630. Scale bar = 10 μm.

D: p16/CD34 coimmunostaining in old-positive glomerular PAI-1 donors. Original magnification X630. Scale bar = 10 μm.

E: Representation of PAI-1 and p16 staining results at time of transplantation in young (n = 8) or old donors (n = 10). Each line represents one patient. Green color is used for negative staining, red color for positive staining.

F: Quantification of the glomerulosclerosis score at time of (M0), or 12 months after (M12) kidney transplantation from elderly donors in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 13) glomerular PAI-1 staining at time of transplantation (M0).

G: Estimated glomerular filtration rate (eGFR) at time of (M0), or 12 months after (M12) kidney transplantation in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 18) glomerular PAI-1 staining at time of transplantation (M0).

H: Urinary PAI-1 measured by ELISA in a cohort of elderly patients with (n = 48) or without (n = 48) chronic kidney disease.

Data information: Data are means ± SEM. Statistical analysis: t-student test for A, B and H, ANOVA followed by the Tukey-Kramer test for F and G.
FTO negatively regulates the cytotoxic activity of natural killer cells (2023)
Figure 1

Figure 1. Loss of Fto expression enhances the anti-metastatic activity of mouse NK cells isolated from spleen.

(A) m6A levels in total RNA from the spleen and bone marrow of Fto+/+ or Fto-/- mice were measured by ELISA (n = 4/group).

(B) Image of the lung surface (left) and numbers of metastatic nodules (right) 14 days following the injection of B16F10 melanoma cells (n = 8/group).

(C) Survival curves after the injection of EL4 lymphoma cells (n = 6/group).

(D, E) Tumor volume (D) and image of the tumor (E) 17 days following the subcutaneous injection (s.c.) of B16F10 melanoma cells (n = 4/group). Tumor size was calculated using the formula length x width2 x 0.5.

(F) Metastatic burden in the lung after treatment with an IgG control antibody (anti-IgG Ab) or anti-NK1.1 antibody for NK cell depletion on days -4, -1, and 2 relatives to B16F10 cell injection (n = 3/group).

(G) EL4 cell killing activity by NK cells isolated from spleen was measured by calcein AM-based assays at the indicated NK: EL4 (E: T, n = 4/group).

(H) Ifng protein and Ifng mRNA levels (n = 4/group) were determined by ELISA and quantitative PCR.

Data information: Error bars represent s.e.m. Significance was determined using the Mann-Whitney U (A, B), Mantel-Cox (C), Two-way ANOVA (D) or Student's t-tests (G, H): ***p < 0.001; **p < 0.01; *p < 0.05.
FTO negatively regulates the cytotoxic activity of natural killer cells (2023)
Figure 2

Figure 2. FTO-deficient NK92 cells display superior killing effects and high cytokine production.

(A) m6A dot blot assay using total RNA from NK92-siCTRL or NK92-siFTO cells.

(B) K562 leukemia cell killing activity by NK92-siCTRL or NK92-siFTO cells was measured by a calcein AM-based cytotoxicity assay at the indicated NK92: K562 (E: T, effector: target) ratio (n = 6 biological replicates).

(C) Degranulation was measured by flow cytometry. Values indicate percentages.

(D) Western blotting for the detection of GZMB in NK92-siCTRL and NK92-siFTO cells after treatment with IL-2.

(E) Levels of IFNγ (n = 5 biological replicates) and TNFα (n = 3 biological replicates) in the culture media 24 hours after IL-2 treatment (left) or co-culture media with K562 cells for 4 hours (right) were determined by ELISA.

Data information: Error bars for panel B and E are ± s.e.m. For panels A, C, and D, data are representative of two (C, D) or three times (A) independent experiments with similar results. Significance was determined using the Mann-Whitney U test (B) or Student's t-tests (E): ***p < 0.001; **p < 0.01; *p < 0.05.
FTO negatively regulates the cytotoxic activity of natural killer cells (2023)
Figure 3

Figure 3. FTO regulates the JAK/STAT signaling pathway.

(A) Western blotting analysis of NK92-siCTRL and NK92-siFTO cells incubated with 20 ng/ml IL-2 for the indicated times was performed. Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated (p) and total proteins (n = 2~3).

(B) Western blotting analysis. NK92-siCTRL or NK92-siFTO cells were incubated with 20 ng/ml IL-2 for 24 hours, washed, and cultured without IL-2 (IL-2 depletion) for the indicated times (n = 3).

(C) A western blotting analysis of NK92+MOCK or NK92+FTO cells incubated with 20 ng/ml IL-2 for the indicated times was performed (n = 3).

Data information: Western blotting data are representative of at least two times independent experiments with similar results. The protein levels were expressed as the relative band density of the corresponding protein (A is on the right, and B and C are on the lower). Error bars are ± s.e.m. Significance was determined using the Student's t-tests: ***p < 0.001; **p < 0.01; *p < 0.05.
FTO negatively regulates the cytotoxic activity of natural killer cells (2023)
Figure 4

Figure 4. FTO controls mRNA stability of SOCS family members by removing m6A RNA modifications.

(A) Relative mRNA expression levels of SOCS family proteins were determined by qPCR after cultivation without IL-2 for 24 hours (n > 8 biological replicates).

(B) Western blotting analysis of SOCS3, CISH, and SOCS1 in NK92-siCTRL or NK92-siFTO cells (n = 3 biological replicates).

(C) m6A methylated RNA immunoprecipitation & quantitative PCR (MeRIP-qPCR) analysis using a m6A antibody in NK92-siFTO (upper) or NK92+FTO (lower) cells compared to NK92-siCTRL or NK92+MOCK cells.

(D) SOCS3 3'-UTR reporter assay using the AANAT reporter system. Plasmid information for the AANAT reporter system with or without the SOCS3 3'-UTR sequence (upper). Levels of Aanat protein (left) and Aanat mRNA (right) expression in HEK-293T cells co-transfected with plasmid-expressing AANAT reporter containing SOCS3 3'-UTR and siCTRL or siFTO.

Data information: Error bars for panel A are ± s.e.m. Error bars for panel C and D are ± s.d. based on technical triplicates. For panels C, and D, data are representative of three times independent experiments with similar results. Significance was determined using the Mann-Whitney U test (A) or Student's t-tests (C, D): ***p < 0.001; **p < 0.01; *p < 0.05.
FTO negatively regulates the cytotoxic activity of natural killer cells (2023)
Figure 5

Figure 5. Activity of FTO-deficient CD3-CD56+ NK cells derived from umbilical cord blood is enhanced.

(A) K562 leukemia cell killing activity by CD56+-siCTRL or CD56+-siFTO cells was measured by calcein AM-based cytotoxicity assays at the indicated NK: K562 (E: T) ratio.

(B) Relative mRNA expression levels of JAK/STAT target genes, IFNγ (n = 10 biological replicates), GZMB (n = 6 biological replicates) and PRF1 (n = 8 biological replicates) were determined by qPCR in CD56+-siCTRL or CD56+-siFTO cells.

(C) Western blotting of CD56+-siCTRL or CD56+-siFTO cells incubated with 20 ng/ml IL-15 and 20 ng/ml IL-21 for the indicated times was performed. Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated (p-) and total proteins (n = 2~3). Data are representative of three times independent experiments with similar results. The protein levels were expressed as the relative band density of the corresponding protein (lower).

(D) Western blotting for the detection of CISH, SOCS1, and SOCS3 was performed in CD56+-siCTRL or CD56+-siFTO cells (n = 3/group).

Data information: Error bars for panel A are ± s.d. based on three technical replicates. Error bars for panel B and C are ± s.e.m. For panels A, data are representative of three times independent experiments with similar results. For panels C, The protein levels were expressed as the relative band density of the corresponding protein on the lower. Significance was determined using the Student's t-tests (A, C) or Mann-Whitney U test (B): ***p < 0.001; **p < 0.01; *p < 0.05.
FTO negatively regulates the cytotoxic activity of natural killer cells (2023)
Figure 6

Figure 6. FTO-deficient NK cells enhance the antitumor responses against K562 leukemia.

(A) Western blotting for the detection of FTO was performed in NK92-TRC or NK92-shFTO cells.

(B) K562-Luc cell killing activity by NK92-TRC or NK92-shFTO cells was measured by calcein AM-based cytotoxicity assay (E: T ratio = 1: 0.625).

(C) Degranulation was measured by flow cytometry (n = 3 biological replicates, E: T ratio = 2: 1).

(D) The mouse experimental scheme. K562-Luc cells were injected, and then NK92-TRC or NK92-shFTO were transfused three times into NOD/Shi-scid/IL-2Rγnull (NOG) mice (n = 6/group).

(E) The images of the ventral bioluminescence (BLI) following the injection of NK92-TRC or NK92-shFTO cells.

(F) The values of the ventral BLI were plotted.

(G) The survival curves after the injection of K562-Luc cells.

Data information: Error bars for panel B are ± s.d. based on three technical replicates. Error bars for panels C and F are ± s.e.m. For panels A, and B, data are representative of three times independent experiments with similar results. Significance was determined using the Student's t-tests (B, C, F) or the Mantel-Cox test (G): ***p < 0.001; **p < 0.01.
Cohesin acetylation and Wapl-Pds5 oppositely regulate translocation of cohesin along DNA (2016)
Figure 1

Figure 1. Cohesin translocates along DNA in an ATPase-dependent manner

(A) Purification of the Halo-tagged cohesin tetramer. Human cohesin tetramer tagged by Halo was purified from insect cells and Alexa488 dye was conjugated to Halo-tag (cohesinHalo488).

(B) CohesinHalo488 loaded onto DNA in the presence of Scc2-Scc4 was incubated in the buffer with or without TEV protease. After high-salt washing, CohesinHalo488 intensities on DNA were measured. Red bars denote the median, lower, and upper quartile values (n ≥ 98; *p < 0.0001, two-tailed Mann-Whitney test).

(C) Kymograph of a single DNA-interacting cohesinHalo488 particle. CohesinHalo488 is loaded onto DNA in the presence of Scc2-Scc4 and, after high-salt washing, single cohesin particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 2 μm.

(D) Histogram of the net displacement (xfinal - xinitial) of cohesin particles treated as in (C). The magenta curve represents the Gaussian fit.

(E) CohesinHalo488 was loaded and washed in high salt as in (C), and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl. Kymograph of DNA-associated cohesin particles under high-salt conditions (750 mM KCl, right) and the mean square displacement (MSD) versus time (left) are shown. Dotted lines are regression lines used to estimate the diffusion coefficient (D). Scale bar, 2 μm.

(F) MSD versus time for DNA-bound cohesinWT-Halo488 or cohesinKA-Halo488 particles. CohesinWT-Halo488 or cohesinKA-Halo488 was loaded onto DNA in the presence of Scc2-4 and ATP. After high-salt washing, the cohesin particles on DNA were observed in the presence of 1 mM AMP-PCP or ATP and 100 mM KCl. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).
Cohesin acetylation and Wapl-Pds5 oppositely regulate translocation of cohesin along DNA (2016)
Figure 2

Figure 2. Acetylation of cohesin antagonizes Wapl-Pds5-dependent suppression of cohesin translocation along DNA

(A) Kymograph of CohesinHalo488 in the presence or absence of Wapl-Pds5. CohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with or without Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 1 μm.

(B) MSD versus time of cohesin particles in (A) are shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).

(C) Acetylated- or unacetylated-cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).

(D) Acetylated cohesinHalo488 was treated as in (C), the DNA-bound cohesin was further incubated with buffer, Wapl-Pds5, or Wapl-Pds5 plus Sororin, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).
Cohesin acetylation and Wapl-Pds5 oppositely regulate translocation of cohesin along DNA (2016)
Figure 3

Figure 3. Plk1- and Aurora B-dependent phosphorylation facilitates cohesin translocation

(A) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5 plus Sororin, and then treated with buffer, Plk1, or Plk1 plus BI4834. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).

(B) Acetylated cohesinHalo488 was treated as in (A), or treated with CDK1 or Aurora B rather than Plk1. Sororin intensity on DNA was measured after immunofluorescence microscopy with anti-Sororin antibody (n > 100, *p < 0.0001, two-tailed Mann-Whitney test).

(C) Acetylated cohesinHalo488 was treated as in (A) but treated with Aurora B rather than Plk1. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).