EMBO/soda-nel huggingface dataset:
Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycetes-specific transcription factor (2021)
Figure 6

Figure 6: Intracellular redox status governs activation of AosR

a. Multiple sequence alignment of AosR from various mycobacterial species. CxxxC motif present within the N terminus extension is highlighted using a blue box. * perfect alignment : strong similarlity and . represents weak similarility.

b. Secondary structure model of AosR predicted by I-Tasser Web server.

c. Early log-phase cultures of indicated strains were subjected to 50 µM of CHP for 24 h and CFUs were enumerated (mean±SEM; n=3). Control represents untreated cells.

d. 1 µM AosR was treated with various ratios of oxidized to reduced DTT (ox:red DTT- 3:1, 2:2, 1:3), or only with 4 µM reduced or oxidized DTT. mBBr was added to the reaction mixture and the fluorescence intensity was measured from 430-600 nm upon excitation at 390 nm wavelength. Reaction mixture without any protein was used as blank (mBBr). Each spectrum represents an average of three individual scans.

e. Early log-phase cultures of indicated strains were subjected to 50 µM of CHP for 24 h and CFUs were enumerated (mean ±SEM; n=3). Control represents untreated cells.

f. pΦ from Balb/C were infected with indicated strains and bacterial viability was monitored 0 and 96 h p.i. Data is presented as mean CFU log10/ml ± SEM and is representative of two biologically independent experiments, each performed in triplicates (n=3).

g. BLI sensorgram illustrating relative binding affinities of 800nM His-AosR and His-AosRAxxxA to immobilized His-SigH under oxidizing conditions.

h. Msm::FLAG-SigH, Msm::FLAG-SigH-AosR-HA and Msm::FLAG-SigH-AosRAxxxA-HA were either subjected to none or 50 µM of CHP for 3 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-HA. 50 µg WCLs were probed with α-FLAG, α-HA and α-SigA as controls.

Data information: Statistical significance was analysed using one-way ANOVA followed by a post hoc test (Tukey test; GraphPad prism). **, p < 0.005; ***, p < 0.0005.

Source data are provided in Source Data file.
Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycetes-specific transcription factor (2021)
Figure 7

Figure 7: AosR is crucial for cellular-redox homeostasis

a. Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified. Data presented is representative of one of two biological replicates, each performed in triplicates (n=3). Y-axis represents µM thiol /µg of WCL (mean ± SD).

b-e. Metabolite levels of reduced mycothiol, MSH (b) ergothioneine, ERG (c) NAD+ (d) NADH (e) were measured in Rv, Rv∆aosR, and Rv∆aosR::aosR in normal and CHP stressed cells using LC-MRM MS/MS analysis. The absolute values obtained under normal conditions were normalized to 100% for each strain and the relative values were calculated for samples processed from CHP treated cells. The data indicates relative levels of metabolites (mean percent ± SD) of four replicates (n=4).

f. To evaluate the contribution of AosR on the antioxidant capacity; Rv, Rv∆aosR and Rv∆aosR::aosR cells were treated with 5 mM H2O2 and the ratiometric response (mean ±SD; n=3) was measured at indicated time points.

g. Schematic depicting the pleiotropic role of AosR. Oxidative environment induces disulfide bond formation in AosR and transcription of mec-cysO-cysM through an auxiliary promoter resulting in enhanced L-cysteine biosynthesis. This further induces genome-wide transcriptome changes. The latter set of changes in indirectly regulated genes can be mitigated by the addition of L-cysteine in the media.

h. qRT-PCR measurements for the log2 fold induction (mean ±SD, n=3) for indicated genes in Rv∆aosR and Rv∆aosR treated with 1mM L-cysteine as compared with Rv. All strains were treated with 50 μM CHP. Data were normalized with respect to 16s rRNA and is representative of one of two biological replicates.

Data information: Statistical significance was analysed using the student's t-test (two tailed, unpaired). *, p<0.05; **, p < 0.005; ***, p < 0.0005, ns = non-significant.

Source data are provided in the Source Data file.
CCG•CGG interruptions in high penetrance SCA8 families increase RAN translation and protein toxicity (2021)
Figure 2

Figure 2. Clustered and interspersed CCG•CGG interruptions increase toxicity of CTG•CAG expansions

A Schematic diagram of constructs used to express patient-derived pure and interrupted SCA8 repeat tracts with predicted protein products and C-terminal epitope tags. * Due to TAG encoded stop codons polyGln proteins do not express an epitope tag. CGG interruptions and the encoded amino acid interruptions are indicated in red.

B, C Cell death measured by lactase dehydrogenase (LDH) (B) and cell viability measured by 3-(4,5- dimethyl-thiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (C) in T98 cells 42 hrs post-transfection of pure and interrupted SCA8 repeat tracts; LDH n=8, MTT n=12, n=independent experiments, * p < 0.05, NT not transfected, EV: empty vector; unpaired t test; mean ± SEM.

D, E qRT-PCR showing transcript levels of Pure 96 and Int.95 (D, n=6; p=0.0187), and Pure 104 and Int.102 (E, n=6; **** p<0.0001) repeats; n=independent experiments; unpaired t test; mean ± SEM.
CCG•CGG interruptions in high penetrance SCA8 families increase RAN translation and protein toxicity (2021)
Figure 3

Figure 3. Arginine interruptions increase toxicity of ATXN8 polyGln proteins

A Alternative-codon constructs for pure and interrupted polyGln assays.

B, C Cell death (B; LDH, n=8) and viability (C; MTT, n=6) assays in T98 cells 42-hrs post-transfection. NT: not transfected; EV: empty vector; n=independent experiments; * p < 0.05; unpaired t test; mean ± SEM.

D qRT-PCR of Alt. polyGln and Alt. Int. polyGln construct transcript levels, n=3 independent experiments; p=0.9516, ns: not significant, unpaired t-test, mean ± SEM.

E-G Protein blots (E) and quantification (F, G) of polyGln proteins expressed in transfected HEK293T cells with interrupted or pure polyGln repeats; EV: empty vector; n=3 transfections, ** p<0.01, *** p<0.001, unpaired t test, mean ± SD.

H Immunofluorescence of polyGln expressed from Pure 104 and Int.102 constructs in HEK293T cells; scale bar: 20μm; EV: empty vector.

I,J Immunofluorescence (I) and quantification (J) of intranuclear inclusions for polyGln expressed from Alt. polyGln and Alt. Int. polyGln constructs in HEK293T cells; scale bar: 50μm; green arrow heads indicate cells positive for Alt. Int. polyGln intranuclear inclusions; dashed white box in merge panels indicates the region show in the orthogonal (Ortho) zoomed images which show cross section and localization of diffuse polyGln staining and nuclear polyGln(Arg) inclusions with alpha tubulin as a cytoplasmic marker; n=4 experiments with n>55 cells per experiment, * p<0.05, **** p<0.0001, two-way ANOVA, data presented as mean percentage of polyGln positive cells ± SD.

K PolyGln(Arg) proteins but not pure polyGln proteins colocalize with nucleophosmin in HEK293T cells; scale bars: 20µm; dashed white box in merge panels indicates the region shown as an orthogonal projection in the ortho zoom panels which show the co-localization of polyGln inclusions and nucleophosmin in three dimensions.
CCG•CGG interruptions in high penetrance SCA8 families increase RAN translation and protein toxicity (2021)
Figure 4

Figure 4. CGG interruptions increase RAN polySer and RAN polyAla protein steady state levels

A-C Protein blotting (A) and quantification (B, C) of polySer RAN proteins in HEK293T cells from interrupted (Int.95; Int.102) or pure (Pure 96; Pure 104) CAG repeats. EV: empty vector, n=3 transfections, ** p<0.01, *p<0.05, unpaired t test, mean ± SEM.

D, E Immunofluorescence (D) and quantification (E) of RAN polySer protein aggregates from CGG interrupted and pure CAG repeat tracts in HEK293T cells; scale bar: 10μm. n>20 cells per construct for each experiment (Exp.), ** p<0.01: Exp.1 p=0.0073, Exp.2 p=0.0017, Exp.3 p=0.0041, Mann-Whitney test; data presented as individual points with median.

F, G Protein blot (F) and quantification (G) of polyAla RAN proteins expressed from pure or interrupted constructs; n=3 transfections, *** p<0.001, unpaired t test, mean ± SD.

H qRT-PCR of Pure 96 and Int.95 transcript levels; n=3 transfections; p=0.9942, unpaired t test, mean ± SEM.

I, J Protein blotting (I) and quantification (J) of phospho eIF2α (p-eIF2α) relative to total eIF2α; n=4 transfections, * p<0.05, unpaired t test, mean ± SD.
Microtubule binding protein Doublecortin-like kinase 1 (DCLK1) guides kinesin-3 transport to dendrites (2015)
Figure 1

Figure 1. Systematic analysis of the transporting properties of kinesin motors

(A) Schematic representation of kinesin constructs used in the inducible trafficking assays. Kinesin constructs contain a motor domain and a coil (MDC).

(B) Principle of inducible peroxisome-trafficking assay. COS-7 cells were transfected with truncated motor constructs of kinesins fused with FRB and GFP (summarized in (A)) and peroxisomes labeled with PEX-mRFP-FKBP. Upon addition of rapalog, kinesin motors are recruited to peroxisomes, and kinesin transporting features can be measured as peroxisome displacement.

(C) Exemplary images of COS-7 cells expressing PEX-mRFP-FKBP before (upper panels) and 30 minutes after (middle panels) rapalog addition in the presence of a translocating motor - KIF1C-MDC-FRB and a non-translocating motor - KIF3B-MDC-FRB. Yellow lines indicate COS7 cell outline. Lower panel represents the overlay of sequential binarized images color coded by time (see also Appendix Figure S1). Scale bar = 20 µm

(D) Plots representing intensity time traces of R90% for COS-7 cells transfected with PEX-mRFP-FKBP and KIF1C-MDC-FRB (n =13) or KIF3B-MDC-FRB (n =10). R90% is the radius for each time point that includes 90% of the total fluorescent intensity. Error bars indicate SEM.

(E) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS-7 cells transfected with kinesin constructs summarized in (A). The number of analyzed cells is summarized in Appendix Table S1. Kinesins were considered translocators, if they displaced peroxisomes ≥ 5 µm (marked with a blue dashed line) in 30 minutes after rapalog addition. Error bars indicate SEM.

(F) Quantitative representation of average dispersion speed of peroxisomes recruited to translocating kinesins as calculated from time traces of R90% (summarized in Appendix figure S2, Appendix Table S1). Error bars indicate SEM.
Microtubule binding protein Doublecortin-like kinase 1 (DCLK1) guides kinesin-3 transport to dendrites (2015)
Figure 2

Figure 2. Kinesin family members display distinct targeting preferences in neurons

(A) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV14 hippocampal neurons expressing PEX-mRFP-FKBP, KIF1C-MDC-FRB, KIF21A-MDC-FRB or KIF5B-MDC-FRB. The morphology of transfected cell is visualized using a BFP fill. Axons are marked with a blue line. Arrows mark peroxisome targeting to dendrites (gray) and axon (blue) 30 min after rapalog addition. Scale bar = 20 µm.

(B) Plots representing intensity time traces of dendrites and axons before and after rapalog addition for KIF1C-MDC-FRB (n = 6 neurons), KIF21A-MDC-FRB (n = 4) and KIF5B-MDC-FRB (n = 8). Traces were normalized to the average intensity before rapalog addition, and to the background.

(C) Quantitative representation of percentage of DIV14 neurons transfected as in (A) with constructs showed in Figure 1A, in which peroxisomes after addition of rapalog redistributed to either axons (”a”, blue bars) or to axons and at least 2 dendrites (”a+d”, red bars). Kinesins were considered ”dendrite targeting” if dendrites were targeted in ≥ 50% of cells and are highlighted in red. Kinesin constructs are transfected as indicated; the number of analyzed cells is summarized in Appendix Table S1. Error bars indicate SEM

(D) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV2 hippocampal neurons transfected as in (A). Scale bars: 20 µm (E) Percentage of DIV2 neurons transfected with KIF1C-MDC-FRB (n=9 neurons), KIF21A-MDC-FRB (n=14) and KIF5B-MDC-FRB (n=14) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog and (F) Percentage of neurites of DIV2 neurons transfected as in (E) targeted with peroxisomes after addition of rapalog. Error bars indicate SEM; Scale bars: 20 µm.
Microtubule binding protein Doublecortin-like kinase 1 (DCLK1) guides kinesin-3 transport to dendrites (2015)
Figure 3

Figure 3. DCLK1 regulates KIF1-mediated transport into the dendrites

(A) Representative confocal images of a hippocampal neuron (DIV14) immunostained for MAP2, β-III-tubulin and DCLK1. Scale bar = 20 µm

(B) Quantification of the average ratio of DCLK1, MAP1A, MAP1B and MAP2 to total tubulin immunofluorescence intensities in dendrites compared to axons. Per condition 6-9 cells were analyzed. Error bars indicate SEM.

(C) Representative images of DIV10 hippocampal neurons transfected for 4 days with plasmids encoding β-gal for visualizing the transfected cells and three different DCLK1 shRNAs or DCLK2 shRNA and immunostained for β-gal (red) and DCLK1 (green).

(D) Quantitative analysis of normalized average intensity of DCLK1 in regions of interest of DIV10 neurons transfected with DCLK1 sh#1 (n = 16 neurons), DCLK1 sh#2 (n=16) and DCLK1 sh#3 (n=18). Non-transfected cells were taken as a control (n = 48). N=2. Error bars indicate SEM; *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).

(E) Example of Western blot analysis of protein extracts of DIV4 cortical neurons electroporated on DIV0 with pSUPER (control) or pSUPER encoding one of three DCLK1 shRNAs. Levels of actin were used as an equal loading control.

(F) Quantification of protein levels of DCLK1 of samples represented in (E). N = 3, error bars indicate SEM, *** - p < 0.001 (1-way ANOVA followed by a Tukey's Multiple Comparison Test).

(G) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX-mRFP-FKBP, KIF1C (MDC)-FRB and the indicated shRNA before and 30 minutes after rapalog addition. BFP fill was co-transected to visualize the morphology of transfected neuron. Axons are marked with a blue line. Arrows mark dendrite (gray) and axon (blue) targeting of peroxisomes after rapalog addition. Scale bar = 20 µm

(H) Quantitative representation of the percentage of DIV14 neurons transfected with PEX-RFP together with either KIF1C, KIF21A or KIF5B and an indicated shRNA. as in (A) in which after addition of rapalog peroxisomes redistributed to axons (”a”, blue bars), to both axons and dendrites (”a+d”, red bars) or did not redistribute into any neuronal compartment (”nt”-no targeting, gray bars). Per condition 19-44 neurons were analyzed; N = 2.
Microtubule binding protein Doublecortin-like kinase 1 (DCLK1) guides kinesin-3 transport to dendrites (2015)
Figure 5

Figure 5. DCLK1 regulates dense-core vesicle entry into the dendrite

(A) Scheme of DCLK1 truncation constructs.

(B) Representative kymographs of dense-core vesicle motility in proximal dendrites of transfected neurons. DIV 10-14 hippocampal neurons were co-transfected for 4 days with NPY-GFP and pSUPER (control) or pSUPER encoding DCLK1 shRNA in the absence or presence of DCLK1 truncation constructs and dense core vesicle motility in dendrites was recorded during live-cell imaging. Scale bar = 5 µm.

(C) Quantification of the average number of dense-core vesicle entries into dendrites of neurons transfected as in (B) during 100 s of the live recording after photobleaching. Per condition 10-32 cells were analyzed. Error bars indicate SEM; *** - p < 0.001 (Kruskal-Wallis test followed by post hoc Dunn’s test). N = 2.

(D, E) DIV10-14 hippocampal neurons were co-transfected for 4 days with either pSUPER (control, n = 20 neurons) or DCLK1 shRNA together with TfR-GFP (n = 16).

(D) Representative kymographs of recycling endosome motility in dendrites. Scale bar = 5 µm. (E) Quantification of the average number of recycling endosome entries into dendrites during 50 s of the live recording after photobleaching. Error bars indicate SEM; ns – not significant (Mann-Whitney test). N = 2.
Microtubule binding protein Doublecortin-like kinase 1 (DCLK1) guides kinesin-3 transport to dendrites (2015)
Figure 6

Figure 6. DCLK1 preferentially binds to a subset of microtubules

(A) Example of Western blot analysis of DCLK1 and DCX levels in protein extracts obtained from DIV1, DIV7, DIV14 and DIV21 hippocampal neurons.

(B) Representative image of a hippocampal neuron (DIV14) immunostained for DCLK1 and β-III-tubulin. Scale bar = 20 µm.

(C, D) Representative image of a COS-7 cell transfected with DCLK1-GFP (C) or DCLK1(ΔKD)-GFP (D) and stained for detyrosinated, tyrosinated or α-tubulin.

(E) Representative image of a COS-7 cell transfected with DCLK1-GFP and TagRFP-EB3 and recorded during live-cell imaging. Representative kymograph was drawn (over a region marked with white boxes) showing that DCLK1-GFP colocalizes with EB3-TagRFP-T. Scale bars = 20 um.
Microtubule binding protein Doublecortin-like kinase 1 (DCLK1) guides kinesin-3 transport to dendrites (2015)
Figure 7

Figure 7. DCLK1 associates with KIF1 and does not affect microtubule polarity in dendrites

(A) Extracts of DIV 7 hippocampal neurons electroporated with pSuper control or with three different DCLK1 shRNAs at DIV0. Samples were analyzed by western blot with indicated antibodies.

(B, E) Representative images of a hippocampal cell transfected at DIV10 with RFP (to visualize transfected cells), pSUPER (control) or DCLK1 shRNA, fixed at DIV14 and stained for EB3. Black lines indicate the neuronal soma outline. Scale bars = 10 µm.

(C, D) Quantification of the number of EB3 comets in the dendrite (C) and in the soma (D). 25-26 neurons were analyzed per condition, N=2. Error bars indicate SEM, *** - p < 0.001, ** - p<0,01 (unpaired t-test).

(F-I) Hippocampal neurons were co-transfected with MACF18-GFP and pSUPER (control) or DCLK1 shRNA and the dynamics of microtubules in dendrites was traced using life-cell microscopy. (F) Representative kymographs of dynamic microtubules ends growth in dendrites with and without photoablation. Scale bar = 10 µm (C). (G, I) Quantification of the fraction of MACF18 events moving retrogradely and anterogradely in a dendrite before (G) and after (I) photoablation. Eighteen neurons were analyzed per condition; N=2. (H) Scheme of live-cell imaging after photoablation.

(J, K) Biotin pull-downs (PD) from extracts of HEK293 transfected with biotin-tagged DCLK1 and (J) KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB, KIF5B-MDC-HA-FRB constructs and probed for HA/DCLK1, (K) different GFP-tagged KIF1A truncation constructs and probed for GFP/DCLK1. The ratio input/pellet is 2% for all pull-down experiments.

(L) GFP pull-down from extracts of HEK293 transfected with DCLK1 fragments and KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB and KIF1C-MD-HA and probed for HA/GFP. LacZ-HA was used as a negative control.
SIRT7 promotes genome integrity and modulates non-homologous end joining DNA repair (2016)
Figure 1

Figure 1 - SirT7-/- mice have increased perinatal lethality and reduced life span.

A Mendelian ratios from SirT7+/- cross (n=522 pups; n=96, 14.5 embryos; P value < 0.001 by 2).

B WT and SirT7-/- mice at 10 days (top) and 2 months (bottom) of age.

C Weight distribution of WT and SirT7-/- mice (n=3-10 female mice per time point and genotype; P value = 0.003 by unpaired t-Test).

D Kaplan Meier survival curves (n=170 WT and n=58 SirT7-/- mice; Log-rank Test P value <0.0001).
SIRT7 promotes genome integrity and modulates non-homologous end joining DNA repair (2016)
Figure 2

Figure 2 - Accelerated aging phenotype in SirT7-/- mice.

A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7-/- mice compared with WT.
B Representative gonadal fat pads from WT and SirT7-/- 16 month old mice.
C Quantitation of body weight (left) and gonadal fat pad mass normalized to total body weight (right) (mean ± SEM; 4 samples per genotype).
D IGF-1 protein levels in serum measured by ELISA (mean ± SEM; 3-8 mice per genotype and age group).
E, F Dot plots of Lin-Sca1+cKit+ (LSK) cells (middle and right) from WT and SirT7-/- bone marrow cells. Cells were gated for negative staining of lineage markers B220, CD3, CD11b, CD19, Gr-1, and Ter-119 (Left), and analyzed for Sca1 and cKit expression (middle and right). (F) Quantitation of (E) (mean ± SEM; 4 mice per genotype).
G Bone marrow, thymus, and spleen cell number in young WT and SirT7-/- mice (mean ± SEM; 3-5 mice per genotype).
H mRNA expression of p16 gene normalized to GAPDH measured by RT-PCR from young and old WT and SirT7-/- fibroblasts (mean ± SEM; 3 samples per genotype).
I, J Senescence-associated β-galactosidase staining (I, blue) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media. (J) Quantitation of the number of senescent cells shown in (I) (mean ± SEM; 3 independent cell lines per genotype).
*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.
SIRT7 promotes genome integrity and modulates non-homologous end joining DNA repair (2016)
Figure 3

Figure 3 - Increased genome instability in SirT7-/- mice.
A, B Dot plots of FACS cell cycle analyses of WT and SirT7-/- MEFs in passage 3 (P3) and 6 (P6) using EdU incorporation and 7AAD (A). Percentages of cells in Sub G1 (apoptotic cells, left square) and cells with DNA content above 4N (polyploid, right square). (B) Quantitation of experiment shown in (A) (mean ± SEM; 3 samples per genotype).
C Survival curve for WT and SirT7-/- thymocytes after X-Ray irradiation (IR) at the indicated doses. Cell death was quantified by FACS using AnexinV and 7AAD staining 18 hours post insult (mean ± SEM; 3 samples per genotype from one of two independent experiments).
D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X-Ray doses, then plated at low density. After 9 days, colonies were stained with crystal violet and counted (mean ± SEM; 3 independent cell lines per genotype).
E Schematic describing the mouse APRT loss of heterozygosity (LOH) assays. Aprt+/- mice were used to measure the in vivo somatic mutation frequency in WT and SirT7-/- mice. Cells fully-deficient for APRT are selected in culture by 2,6-diaminopurine (DAP), an adenine analog that is converted to a toxic product by APRT enzymatic activity. Mutant frequency is proportional to the number of DAP-resistant (DAPr) colonies.
F Quantitation of experiment shown in (E) (mean ± SEM; 9 WT and 3 SirT7-/- samples).
*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.