EMBO/soda-nel huggingface dataset:
SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation (2021)
Figure 2

Figure 2 - SIRT7 interacts with, stabilizes and increases KCC4 activity and protein levels.

A, B KCC4 activity (A), and protein levels (B) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD+. Non-injected oocytes (UI) were used as a control.

C KCC4 activity was evaluated in Xenopus oocytes injected with cRNA encoding KCC4 and coinjected with either wild-type (SIRT7 WT) or the catalytically inactive SIRT7 (SIRT7 H188Y) for 48 h.

D Oocytes were injected with either KCC4 WT, K114R or K114Q for 48 h and then incubated with vehicle (V), NAD+ or NAM for 24 h, and KCC4 and SIRT7 protein levels were analyzed by SDS-PAGE/immunoblotting.

E Oocytes were injected with either KCC4 WT, K114R or K114Q with and without SIRT7 for 48 h and then incubated with vehicle (V) or NAM for 4 h. After lysis, KCC4 was immunoprecipitated using an anti-KCC4 antibody, and the acetylation status of KCC4 was determined by SDS-PAGE/Western blotting using and anti-acetyl lysine antibody.

F, G FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ (F) or NAM (G) and then analyzed by SDS-PAGE/immunoblotting using an anti-KCC4 or anti-SIRT7 antibody. Non-transfected cells (UT) were used as a control.

H FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ and NAM. After lysis, KCC4 immunoprecipitation using an anti-FLAG antibody was performed, and the interaction with SIRT7 was determined by SDS-PAGE/Western blotting.

I HEK293 cells expressing KCC4 were incubated with NAD+ 200 μM and cycloheximide (CHX) for 0, 3, 6 and 9 h. KCC4 protein levels are shown at the indicated times in cells transfected with a scramble shRNA (+SIRT7) or a shRNA against SIRT7 (-SIRT7).

Data information: Data are presented as mean ± SEM. Depicted blots are representative of three independent biological replicates and values shown are the mean of the three experiments. To evaluate KCC4 activity, we performed three independent biological experiments with oocytes obtained from different frogs and each experiment included at least 4 replicates. *Indicates a significant difference vs time 0 or vs KCC4 WT or vs +SIRT7 at p<0.05 by Student's t-test. #P<0.05 when compared to Vehicle by Student´s t-test.
SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation (2021)
Figure 3

Figure 3 - SIRT7 regulates KCC4 activity during a decrease in external pH.

A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4. After lysis, KCC4 was immunoprecipitated using an anti-FLAG antibody, and then analyzed by SDS-PAGE/Western blotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-transfected cells (UT) were used as a control.

B HEK293 cells were transfected with KCC4 together with SIRT7 with a scramble shRNA (-) or a shRNA against SIRT7 (+), and KCC4 activity and protein level were determined at the indicated pH. SIRT7 expression in these conditions is presented in C.

C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells. At a lower pH, SIRT7 appears to translocate to the cytoplasm.

Data information: Data are presented as mean ± SEM. Depicted blots are representative of three independent biological experiments and values shown are the mean of the three experiments. To evaluate KCC4 activity, we performed three independent biological experiments and each experiment included at least 3 replicates. *Indicates a significant difference vs pH=7.7 at p<0.05 by Student's t-test. # Indicates a significant difference vs KCC4 at pH=6.2 at p<0.05 by Student's t-test.
SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation (2021)
Figure 4

Figure 4 - Characterization of renal SIRT7 expression in C57BL6 mice.

A, B Sirt7 mRNA (A) and protein (B) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau, respectively. Enrichment for each segment was previously validated by immunoblotting against segment-specific markers as indicated in material and methods (Spirli et al, 2019).

C Immunolocalization of SIRT7 and ATPase in the kidneys of C57BL6 mice with metabolic acidosis (SIRT7, red; ATPase, green; DAPI, Blue). Scale bar: 20 μm. Controls of the specificity of antibodies using kidney of SIRT7-deficient mice are provided in Appendix Fig S4.

D KCC4 and SIRT7 protein levels in kidneys from control mice or mice with metabolic acidosis. Values are the mean ± SEM.

Data information: Data are presented as mean ± SEM. For each segment, tubules isolated from different animals were pooled (PT n=3, TAL n=5, DCT/CNT n=5 and CCD n=5). The western blot was performed with the pooled samples and repeated to assure reproducibility. Each lane of the western blot depicted in D is a biological replicate as each lane represent one mouse. The blot was repeated with three other mice and the quantification showed is the average of the seven mice. *Indicates a significant difference vs control at p<0.05 by Student's t-test.
SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation (2021)
Figure 5

Figure 5 - KCC4 protein levels in the kidneys and metabolic acidosis status of SIRT7-deficient mice and renal tubule-specific SIRT7-deficient mice.

A KCC4 protein levels were determined in the kidneys of wild-type (SIRT7+/+) and SIRT7-deficient mice (SIRT7-/-). SIRT7 protein expression are absent in the kidneys of SIRT7-deficient mice.

B KCC4 protein levels, acetylation status of KCC4, and interaction with SIRT7 in kidneys of SIRT7+/+ and SIRT7-/-mice under normal conditions (Control) or with metabolic acidosis.

C Immunolocalization of KCC4 and H-ATPase in the kidneys of SIRT7+/+ and SIRT7-/- mice under normal conditions (Control) or with metabolic acidosis (NH4Cl). (KCC4, red; H-ATPase, green). Scale bar: 20 μm. Cell count was performed automatically using the Gen5 software to report the percentage of KCC4+ cells among H-ATPase+ cells (lower panel).

D NCC and NKCC2 protein levels in the kidney of SIRT7+/+ and SIRT7-/- mice under basal conditions.

E Scheme illustrating the proposed mechanism by which SIRT7 regulates KCC4 in the kidney. Briefly, KCC4 maintains a low intracellular Cl- concentration to ensure that basolateral Cl-/HCO3- exchange remains active to allow extrusion of HCO3- from the cell, specifically in α-intercalated cells of the collecting duct, and therefore warranting a constant production of H+ that are secreted through the apical membrane to be eliminated in the urine. During stress, such as metabolic acidosis, SIRT7 levels increase in the cytoplasm, promoting the interaction and stability of KCC4. This interaction induces an increase in KCC4 protein levels and activity and therefore contributes to the increase of basolateral Cl- efflux, which decreases the intracellular Cl- concentration, thus maintaining the gradient for the Cl-/HCO3- exchanger to eliminate bicarbonate from the cells and increase the secretion of H+ to reestablish acid-base homeostasis.

Data information: Data are presented as mean ± SEM. Depicted blots are representative of two different western blot analysis performed with six mice in total. The values presented are the mean of the seven mice. Depending on availability actin, tubulin or GAPDH were used as control. * Indicates a significant difference vs SIRT7+/+ at p<0.05 by Student's t-test.

Expanded View Figure legends
Glutathione peroxidase activity is neuroprotective in models of Huntington's disease (2013)
figf1

(a) Schematic representation of the genome-wide systematic ORF suppressor screen in yeast. (b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF. (c) Network analysis showing a high degree of interconnectivity among the ORF suppressors. The network data were analyzed with an iterative minimum filter of 2. The functional groups are color coded as indicated in the figure. A total of 57 nodes with 115 edges define this network.
Glutathione peroxidase activity is neuroprotective in models of Huntington's disease (2013)
figf2

(a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression. Overexpression of mGpx1 increases total GPx activity in PC12 cells (****P 0.0001 compared to uninduced controls; n = 7 per condition). (b) Quantification of caspase-3/7 activation in response to Htt103Q expression in cells overexpressing mGpx1. Overexpression of mGpx1 reduces Htt103Q-mediated caspase-3/7 activation (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q (n = 12 (WT); n = 9 (monomeric red fluorescent protein (mRFP)); n = 8 (mGpx1) per condition). (c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition). (d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.
Glutathione peroxidase activity is neuroprotective in models of Huntington's disease (2013)
figf3

(a) Pseudopupil images from wild-type flies, Huntington's disease flies and Huntington's disease flies overexpressing mGpx1 at day 10. In wild-type flies, seven rhabdomeres are visible per ommatidium. Scale bar, ∼10 μM. (b) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with and without pan-neural mGpx1 overexpression at days 1 (n = 14 (Htt93Q); n = 9 (Htt93Q + mGpx1)), 7 (n = 7 (Htt93Q); n = 9 (Htt93Q + mGpx1)) and 10 (n = 8 (Htt93Q); n = 9 (Htt93Q + mGpx1)) after eclosion. Significant effects of genotype (F1,50 = 82.3, P = 3.82 × 10−12) and age (F2,50 = 94.3, P = 1.06 × 10−17) were observed, as well as a significant interaction between genotype and age (F2,50 = 7.76, P = 0.00116), indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies. Post-hoc Newman-Keuls comparisons showed significant rescue of neurodegeneration in mGpx1-expressing flies at days 1 (*P 0.05), 7 (***P 0.001) and 10 (***P 0.001). Browne-Forsythe analyses for equality of variance revealed a significant inequality of variance (P = 0.02) due to a single group (Htt93Q + mGpx1, day 1). The nonparametric Mann-Whitney test confirmed a difference at day 1 between the two groups (P = 0.03), and between Htt93Q + mGpx1 flies at days 1 and 7 (P 0.0001). (c) Total locomotor activity was assessed as the average number of beam crossings per 30-min bin per fly. Htt93Qex1-expressing flies (n = 29) have reduced locomotor activity compared to controls (***P 0.001; n = 30 (WT (w118)); n = 32 (mGpx1)), which is restored by mGpx1 (**P 0.01; n = 32). Comparisons were performed by Kruskal-Wallis one-way ANOVA. (d) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7. Flies expressing Htt93Qex1 driven by Pdf-GAL4 (n = 16 brain hemispheres) showed loss of s-LNVs compared to Canton S (CS) control flies (n = 13), which was ameliorated by coexpression of mGpx1 (****P 0.0001; n = 16). Comparisons were performed by Kruskal-Wallis one-way ANOVA. (e) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with ebselen or vehicle (DMSO; n = 10) treatment at day 7 after eclosion and in newly emerged day 0 adult flies (untreated control; n = 10). Significant treatment effects were observed, both with the 100 μM (**P 0.01; n = 9) and 300 μM (***P 0.001; n = 9) doses of ebselen. All data are shown as the mean ± s.e.m.
Glutathione peroxidase activity is neuroprotective in models of Huntington's disease (2013)
figf4

PC12 cells were treated with either 10 μM ebselen or 10 mM N-acetylcysteine (NAc) for 24 h, with the addition of 400 nM bafilomycin A1 for the final 4 h. Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h. (a) Autophagy was determined by measuring the amount of LC3-II by immunoblot analysis. (b) Densitometric analysis of the bands was performed using ImageJ, and the amounts of LC3-II are expressed relative to the untreated control. All statistical analyses were performed using one-way ANOVA with post-hoc tests (****P 0.0001). All data are shown as the mean ± s.e.m.; n = 7 per condition.
Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage (2022)
Figure 2

Figure 2. Role of ATP in condensin restraining of negative DNA supercoils

(A) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were incubated at 30ºC for indicated time periods (0 to 90 min) in the presence of different concentrations of ATP (1, 0.25, 0.1 mM).

(B) Plot of ∆Lk values (mean ±SD, 3 technical replicates) constrained in the preceding experiment.

(C) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 10 min (lane 1), with ATP 1mM for 10 min (lane 2), AMPPNP 2 mM for 10 min (lane 3), and AMPPNP 2mM for 10 min followed by ATP 1 mM for 10 min (lane 4).

(D) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 10 min (lane 1), with Apyrase for 10 min (lane 2), Apyrase and ATP 1mM for 10 min (lane 3), ATP 1mM for 10 min (lane 4), no nucleotide for 10 min followed by Apyrase for 60 min (lane 5), ATP 1mM for 10 min followed by Apyrase for 60 min (lane 6).

(E) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 20 min (lane 1), with ATP 1mM for 20 min (lane 2), AMPPNP 2 mM for 20 min (lane 3), ATP 1mM for 10 min followed by AMPPNP 2 mM for 10 min (lane 4).

(F) 2D-gel of the samples in E (lanes 1 to 4) and histogram of Lk intensities (i) of lanes 1 and 4, indicating Lkº, LkC and ∆Lk.

Data information: DNA electrophoreses were conducted and labelled as in Fig 1.
Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage (2022)
Figure 3

Figure 3. Endonuclease assays to test DNA unwinding by condensin

(A) DNA unwinding (∆Tw<0) restrains negative ∆Lk.

(B) Left-handed bending or looping of DNA (∆Wr<0) restrains negative ∆Lk.

(C) Negatively supercoiled (0.3 nM) and relaxed DNA (0.3 nM) were incubated with/without nuclease P1 for 10 min at 30ºC in condensin reaction buffer.

(D) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I were mixed with/without nuclease P1 and incubated at 30ºC with no nucleotide for 30 min, ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min.

(E) Experiment conducted as in D, but using (-) supercoiled DNA and without Topo I. Following the incubation with nucleotides for 30 min, nuclease P1 was added for 5 min.

Data information: Supercoiled (S), relaxed (R), nicked (N) and linear DNA signals are indicated.
Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage (2022)
Figure 5

Figure 5. DNA supercoiling does not spread outside the condensin-DNA complex

(A) Either the DNA-strand rotation mechanism of Topo I or the DNA-cross inversion mechanism of Topo II could relax compensatory (+) supercoils outside the condensin-DNA complex.

(B) Relaxed DNA (0.3 nM) and condensin (3 nM) were mixed with Topo I or Topo II (1 unit). Upon addition of ATP (1 mM), incubations proceeded at 30ºC for 30 min.

(C) DNA, condensin, and Topo II were mixed as in B. Upon addition of ATP (1 mM), incubations proceeded at 30ºC for 10 min. Topo I was added to one half of the mixtures and incubations continued for 10 min.

(D) DNA, condensin, and Topo II were mixed as in B. Following incubation at 30ºC for 10 min with or without ATP, DNA was recovered and nicked to reveal the occurrence of DNA knots. K3 to K6 denote knots with 3 to 6 irreducible DNA crossings.

(E) The presence of a nick would relax the compensatory (+) supercoils outside the condensin-DNA complex; and subsequent nick ligation would capture the restrained (-) supercoils.

(F) Nicked DNA (0.3 nM) was mixed without or with condensin (3 nM) in the presence of ATP (1 mM). Following incubations at 30ºC for 20 min, mixtures were supplemented with T4 DNA Ligase and Topo I (as indicated) and incubation continued for 10 min.

(G) If two DNA binding sites delimit a short topological domain, which is subsequently pinched into a (-) supercoil, the compensatory (+) supercoiling would remain within such topological domain,
Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage (2022)
Figure 6

Figure 6. Condensin does not generate twin supercoiled loops while restraining negative supercoils.

(A) If condensin translocation produces twin (+) and (-) supercoiled domains, (+) supercoils would accumulate upon relaxation of (-) supercoils with TopA.

(B) Relaxed DNA (0.3 nM) was mixed with condensin (3 nM). Following incubation in absence or presence of the indicated nucleotides (ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min), TopA was added to one half of each reaction and Topo I to the other half. Incubations continued for 10 min.

(C) Relaxation of unconstrained (-) supercoils by either Topo I or TopA could reveal condensin ATP-dependent stabilization of pre-existing (-) supercoils.

(D) Experiment conducted as in B but starting with (-) supercoiled DNA instead of relaxed DNA.

Following incubation in presence of the indicated nucleotides (as in B) Topo I was added to one half of each mixture and incubations continued for 10 min.

(E) Experiment conducted as in D but adding TopA instead of Topo I.
Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage (2022)
Figure 7

Figure 7. Ycg1 subunit is not required to restrain negative supercoils

(A) Relaxed DNA (0.3 nM), ∆Ycg1 condensin (3 nM) and Topo I were incubated at 30ºC with no nucleotide for 30 min, ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min. The plot (mean ±SD, 3 technical replicates) compares the ∆Lk values restrained by the condensin holo-complex and the ∆Ycg1 condensin.

(B) Salt resistance of the ∆Ycg1 condensin-DNA complex. Experiments were conducted as in Fig 4D-H. The plot (mean ±SD, 3 technical replicates) compares the fractions of DNA bound to the holo-complex and to the ∆Ycg1 tetramer.
The 18S rRNA m6A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation (2020)
Figure 1

Figure 1. METTL5 is the N6-adenosine methyltransferase targeting 18S rRNA in vivo and in vitro.

A Schematic diagram of sgRNA targeting mouse Mettl5 locus. sgRNA targeting site is highlighted in red. PAM sequence is underlined and highlighted in green.

B Western blot analysis confirming two Mettl5 KO clones by specific METTL5 antibody. α-TUBULIN was used as a loading control.

C LC-MS/MS analysis of m6A/A levels in purified 18S rRNA and 28S rRNA from the WT and Mettl5 KO (KO-6B) mESCs.

D PAGE gel electrophoresis showing the PCR amplification of the elongated and ligated products of SELECT method for detecting m6A1832 site and A1825 site (for input control) in 18S rRNA of WT and Mettl5 KO (KO-6B) mESCs, respectively.

E LC-MS/MS analysis of d3-m6A/A levels in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 proteins in vitro. No enzyme group was used as a negative control.
The 18S rRNA m6A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation (2020)
Figure 2

Figure 2. Characterizations of the Mettl5 KO mESCs.

A,B Representative bright-filed (A) and AP staining (B) images of the WT and Mettl5 KO mESCs cultured in 2i/Lif medium. Scale bars represent 100 μm and 50 μm, respectively.

C RT-qPCR analysis of the expression of pluripotency genes in WT and Mettl5 KO mESCs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; ***, P<0.001.

D Western blot analysis of the expression of pluripotency markers in WT and Mettl5 KO mESCs. α-TUBULIN was used as a loading control.

E Immunostaining analysis of the expression of pluripotency markers (POU5F1, SOX2 and NANOG) in WT and Mettl5 KO mESCs, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 10 μm.
The 18S rRNA m6A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation (2020)
Figure 3

Figure 3. Mettl5 KO impairs early differentiation of mESCs.

A Western blot analysis showing dynamic expression patterns of POU5F1 and METTL5 during the indicated time points of EB induction. α-TUBULIN was used as a loading control.

B Histogram showing the threshold cycles of qPCR (qPCRCT) for detecting m6A1832 site in 18S rRNA during the indicated time points of EB induction. Data are shown as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *, P<0.05.

C Immunostaining analysis of the expression of pluripotency marker POU5F1(left), endoderm marker FOXA2 (middle) and neuroectoderm marker NESTIN (right) in control and Mettl5 KO EBs at different time points, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 100 μm.

D Quantification of the percentages of positive cells shown in (C). Quantitative analysis was based on at least three independent experiments. Data are represented as mean ± SD. Student's t test, two-tailed. **, P < 0.01; ***, P<0.001.

E RT-qPCR analysis of the expressions of pluripotency and lineage-specific markers in Day7 EBs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P<0.001.

F Western blot analysis showing pluripotency (POU5F1) and three germ layer markers (FOXA2, T and NESTIN) expression in Day7 EBs. α-TUBULIN was used as a loading control.