EMBO/soda-nel huggingface dataset:
Molecular determinants of selective clearance of protein inclusions by autophagy (2012)
f6

(a–d) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin-1 (a), Vps34 (b), Atg14L (c) and DFCP-1 (d). Arrows indicate colocalization.
Molecular determinants of selective clearance of protein inclusions by autophagy (2012)
f7

(a) Quantitative FRAP analysis of the indicated GFP-p38 variants. Top: representative images. Middle: time course of changes in relative fluorescence intensity (RFI). Bottom: percentage of proteins in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Scale bars, 10 μm (b) Top: schematic showing all lysine (K) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site(s). Bottom: relative fold difference in the number of Agm formed by WT and the different ‘K to R’ mutants of ANK1-p38 fusion protein in SY5Y cells maintained in the presence or absence of serum. (c) IBs for HA (top) and GFP (bottom) of total lysates (Input) and GFP immunoprecipitates (IP) from the cell lysates of SY5Y cells co-transfected with the indicated HA–ubiquitin constructs and GFP-p38 or -ANK1-p38 WT or mutant K385R in the presence of lactacystin. (d) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination. Values were corrected by levels of GFP immunoprecipitated. (e) Top: IF for GFP and K63-linked ubiquitin (red) of SY5Y cells expressing GFP-p38 or -ANK1-p38 WT and mutant K385R (green). Arrows indicate colocalization. Bottom: percentage of Agm positive for K63-linked ubiquitin. (f) Quantitative FRAP analysis of GFP-ANK1-p38 K385R and GFP-ANK1-p38 as in (a). Values are mean+s.e. of ten cells analysed over three different experiments. (g) Top: representative images of GFP-ANK1-p38 K385R Agm in SY5Y cells immunostained for Atg14L, Vps34 and DFCP-1. Bottom: percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L, Vps34 and DFCP-1 under serum− conditions. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition or p38 Agm. #Significant versus ANK1-p38 Agm. Significance calculated using Bonferroni test. See also Supplementary Fig. S8.
Molecular determinants of selective clearance of protein inclusions by autophagy (2012)
f8

(a,b) Autophagic clearance of Agm (a) or Agg (b) formed by FL WT and mutant K385R or K394R GFP-Sph1 in SY5Y cells, in response to serum removal (a) or upon blockage of basal autophagy with 3-MA (b). (c,d) Left: Agm (c) or Agg (d) of FL GFP-Sph1 (green) immunostained for endogenous K63-linked ubiquitin (red) in SY5Y cells. Arrows in (c) indicate colocalization. Right: percentage of Agm (c) or Agg (d) positive for K63-linked ubiquitin. (e) Formation of WT and mutant K385R FL Sph1 (left) and ANK1-p38 (right) Agm in response to proteasome inhibition. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,b) or WT FL Sph1 or ANK1-p38 (c–e). (f) Schematic model of the contribution of the ANK1 region and K63 linkage of this region to removal of Agg and Agms through different types of autophagy.
Histone crotonylation regulates neural stem cell fate decisions by activating bivalent promoters (2021)
Figure 4

Figure 4. Histone Kcr activates bivalent promoters to stimulate gene expression.

A ChIP-seq density heatmaps in NSPCs for histone Kcr, H3K27me3, H3K4me3, and H3K27ac at ± 1 kb (histone Kcr), ± 2.5 kb (H3K4me3 and H3K27ac), and ± 5 kb (H3K27me3) from the center of 1447 peak regions located in bivalent promoters with increased histone Kcr after crotonate treatment.

B Boxplot showing expression changes of 1101 genes that were annotated in the 1447 peak regions defined in (A). Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.001.

C GO analysis (biological process) of the 1101 genes defined in (B).

D BETA plot of combined computational analysis of histone Kcr ChIP-seq and RNA-seq data, peak regions located in bivalent promoters with increased histone Kcr as input.

E Quantification analysis of changes in histone Kcr levels in NSPCs treated with DMSO (Control), 5 μM triptolide (FP), 5 μM flavopiridol and 10 mM crotonate (FP+CR), and 10 mM crotonate (CR) for 1 h. Data are presented as mean ± SEM of three independent biological replicates, n = 3. One-way ANOVA with post hoc Tukey's test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001.

F Co-IP analysis indicating RNAP2 binding in H3K9cr enriched regions in NSPCs. H3 was used as a loading control for the input nuclear proteins.

G Quantification analysis of changes in chromatin openness at Tgfb1 and Notum promoter regions by FAIRE-qPCR among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05, P < 0.01.
Histone crotonylation regulates neural stem cell fate decisions by activating bivalent promoters (2021)
Figure 5

Figure 5. Crotonate alters NSPCs fate via transcriptome remodeling.

A, B Representative images (A) and quantification analysis (B) of changes in cell proliferation ability measured by BrdU incorporation rate among different groups of NSPCs. Scale bar, 50 μm. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.01.

C, D Representative images (C) and quantification analysis (D) of changes in neuronal differentiation potential defined by TUJ1 positivity among different groups of NSPCs. Scale bar, 20 μm. Data are presented as mean ± SEM of three independent biological replicates, n = 4. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.001.

E, F Odds-ratio analysis of overlapping genes displaying different expression (DEx) versus representing changes in cell proliferation ability (E) and neuronal differentiation potential (F) in NSPCs. Np: Non-proliferative cells, and P: proliferative cells. Up and Down: upregulated and downregulated genes after crotonate treatment in NSPCs, respectively. Insert numbers indicate respective P-values for associations, with the number of genes overlapping in parentheses, and two-sided Fisher's exact test was used to analyze statistical significance.

G Genome-browser view at the Mir-203 gene of different sequencing datasets, with annotation of chromatin state at the top.

H Quantification analysis of changes in histone Kcr at Mir-203 proximal regions by ChIP-qPCR among different groups of NSPCs. The amplified genomic regions of two pairs of primers (P1 and P2) are shown in (G). Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05, P < 0.01.

I, J Quantification analysis of expression changes of Mir-203 (I) and Bmi1 (J) by qRT-PCR among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05, P < 0.01.

K Representative blots (top panel) and quantification analysis (bottom panel) of changes in BMI1 protein levels among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05.

L Venn diagram of a combined comparison of different gene sets.

M, N GO analysis (biological processes) of overlapping genes in (L) between red circle with blue circle (M), and red circle with purplish red circle (N).
Histone crotonylation regulates neural stem cell fate decisions by activating bivalent promoters (2021)
Figure 6

Figure 6. Regulation and dynamic changes of histone Kcr in vivo.

A, B Core histones were prepared from E13.5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti-H3K9cr (A) and anti-Kcr (B) antibodies. Naïve: isolated forebrains without subsequent culture and treatment.

C, D Quantification analysis of changes in H3K9cr (C) and H3-Kcr (D) levels by western blotting among different groups of explants. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001.

E, F GO analysis (biological process) of genes with increased (E) and decreased (F) expression levels after crotonate treatment in E13.5 forebrain explants.

G, H Quantitative analysis of changes in H3K9cr (G, left panel of H) and H3K14la (right panel of H) levels by western blotting among different groups of the embryonic forebrain. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; NS: no significance, P < 0.05, P <0.001.

I Quantitative analysis of changes in H3K9cr levels in the developing forebrain. Data are presented as mean ± SEM of three independent biological replicates, n = 3. One-way ANOVA with post hoc Tukey's test was used to analyze statistical significance; NS: no significance, P < 0.05, P <0.01.
Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors (2018)
Figure 1

Figure 1.

PKCδ restricts HSPC pool size in the bone marrow.

A. Quantitative real-time PCR analysis of PKCδ mRNA levels in FACS sorted Lin-, LT-HSC, ST-HSC, MPP, L-S-K+, GMP, CMP, MEP and CLP subsets from C56BL/6 wild-type (6-to 9-week old) mice bone marrow. Levels of PKCδ expression were normalized to an internal control gene (β-actin). Expression of PKCδ is shown relative to Lineage negative (Lin-) cells whose expression was arbitrarily set to 1 (n=4 mice analyzed for each population).

B, C (B) FACS plots depict the percentage of live, Lin-Sca1+c-Kit+ (LSK) (left panel), and primitive CD34-Flt3-, CD34+Flt3- and CD34+Flt3+ HSPCs among LSK cells (right panel). (C) Bar charts show the average frequency (left) and absolute number (right) ± SEM of the indicated populations, analyzed from 2 femurs and 2 tibias per mouse (n=10 mice analyzed for each genotype).

D. FACS plots show percentages of 'SLAM-code' based stem and progenitor cells in the LSK population. Bar graphs show the average frequency (left) and absolute numbers (right) ± SEM of LT-HSC per 2 femurs and 2 tibias. (n=10 mice analyzed for each genotype).

E. FACS plots illustrate gating and frequencies of myeloid progenitors (MP), granulocyte-monocyte progenitor (GMP), common myeloid progenitor (CMP), and megakaryocyte-erythroid progenitor (MEP) population. Bar graphs show the frequencies (left) and absolute numbers (right) of L-S-K+ MP and GMP, CMP and MEP populations in the BM of WT (n=12) and PKCδ KO (n=12).

F. FACS plots illustrate the gating and percentages of common lymphoid progenitors (CLP). Bar graph shows the absolute number of CLPs per 2 femurs and 2 tibias (n=5 mice for each genotype).

G. FACS plots illustrate the percentages of uncommitted lymphoid progenitors (Lin- IL-7Rα+Flt3+Ly-6D-) and B lymphoid progenitors (BLP). Bar graph represents the average frequencies of CLPs, BLP and uncommitted lymphoid progenitors in total BM (n=5 mice per genotype).

H. Limiting dilution analysis (LDA) demonstrates an increased frequency of long-term repopulating HSCs in PKCδ KO BM (solid red lines) compared with WT BM (black solid lines). Engraftment data shown at 14-weeks post-BMT. Plots depict the percentages of recipient mice containing less than 1% CD45.2+ blood nucleated cells. Dotted lines represent the 95% confidence interval of the same (p=0.0005).

Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with Sidak's multiple comparisons test (A) or two-tailed Student's unpaired t-test (C-G) analysis for comparison of WT to PKCδ KO mice. Overall test for differences in stem cell frequencies between WT to PKCδ KO mice was determined with likelihood ratio test of single-hit model (H).
Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors (2018)
Figure 2

Figure 2.

Accelerated proliferation and reduced apoptosis in subsets of PKCδ-deficient HSPCs.

A. Representative FACS profiles of HSPC cell cycle analysis using combinatorial staining for Ki67 and Hoechst 33342. Bar charts depict the average percentage of cells in each phase of the cell cycle for each LSK subset from WT (n=6) and PKCδ KO (n=7) mice. Data compiled from two independent experiments.

B. FACS profiles of indicated BM subsets from WT and PKCδ KO mice 20hr after BrdU injection.

C. Average percentages of cells in each phase of the cell cycle phases for each of the indicated HSPC subsets from WT and PKCδ KO mice. Data are pooled from two independent experiments (totaling n=6-7 mice per genotype).

D. Representative FACS plots and summary of FACS data analyzing the frequency of apoptotic HSPC cells using co-staining for AnnexinV and 7-AAD (WT and PKCδ−/− (n=7 per genotype).

Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.
Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors (2018)
Figure 3

Figure 3.

Loss of PKCδ enhances competitive repopulation and self-renewal of HSPCs in vivo without exhaustion.

A. Schematic of competitive BM transplantation assay.

B. Percent of total donor-derived, hematopoietic cells (CD45.2+), B-cells (B220+), myeloid cells (CD11b+Gr1+), and T cells (CD3+) in the peripheral blood (PB) of recipient mice, as determined by FACS at the indicated time points. The statistical significance of differences was determined using two-ANOVAs with Holm-Sidak's multiple comparisons tests (n=8 recipients per genotype in each experiment).

C. Percent donor-derived HSPCs in the BM of secondary (left) and tertiary (right) recipients of WT or PKCδ−/− marrow. Data are compiled from two independent experiments (n=8 recipients per genotype).

Data information: All data are presented as mean±SD. *p<0.05, **p<0.001, with significance determined by repeated measures of two-way ANOVA analysis with Sidak's multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population.
Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors (2018)
Figure 4

Figure 4.

Augmented HSPC pool size following loss of PKCδ in hematopoietic and stromal cells.

A. Experimental design for pIpC treatment and analysis of PKCδ fl/fl/Mx-1Cre- (PKCδ fl/fl) and PKCδ fl/fl/Mx-1Cre+ (PKCδ Δ/Δ, cKO) mice.

B,C Representative FACS plots of PKCδ fl/fl and cKO BM 4-8 weeks (B), and 20-24-weeks (C) after pIpC treatment. Numbers in plot indicate the absolute numbers of the indicated subset within the gated population (n=8-9 mice per genotype and time point).

D. Schematic of competitive BM transplants. Total BM cells from either PKCδ fl/fl/Mx-1Cre- (control) or PKCδ fl/fl/Mx-1Cre+ mice were injected together with recipient type (CD45.1) BM cells (1:1) into lethally irradiated CD45.1 mice prior to pIpC treatment. Two months after transplant, both groups of mice received five doses of pIpC. Chimeras were analyzed at 16-weeks after the last pIpC injection.

E, F (E) FACS plots show contribution of donor-derived (CD45.2+) HSPCs in recipient BM (E) Calculated absolute numbers of donor-derived HSPC subsets in transplanted mice (F). (total of n=7-8 mice per genotype).

G. Average percentage BrdU+ HPSCs (3 days after BrdU injection into mice reconstituted 24 weeks previously with control or cKO marrow and injected 16 weeks previously with pIpC, see panel (D) among donor-derived LSK cells in the control and cKO chimeras BM.

H. Percentages of apoptotic cells (AnnexinV+/7-AAD-) in donor-derived LSK cells in control and cKO chimera mouse BM at 24 weeks after transplant and 16 weeks after pIpC injection. Data are compiled from two independent experiments (total of n=7-8 mice per genotype).

Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.
Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors (2018)
Figure 6

Figure 6.

Increased mitochondrial number, activity, energy metabolism and ROS levels in PKCδ deleted HSPCs.

A. MitoStress test revealed increased oxygen consumption rates (OCR) in PKCδ-deleted LSKs. BM LSK cells were sorted from WT and PKCδ cKO mice 12-weeks after pIpC-treatment. OCR was measured at basal level and after sequential loading of ATP synthase inhibitor Oligomycin (350nm), mitochondrial uncoupler, FCCP (10μM), and electron transport chain inhibitor, Rotenone (1μM) using Seahorse XF24 extracellular flux analyzer. Data are from 3 independent experiments (n=9 mice). In each experiment, bone marrow cells from identical genotypes (n=3 mice per genotype) were pooled and used for LSK cell sorting.

B, C (B) Basal OCR was measured before inhibitors treatment (left); and the Maximal OCR capacity after FCCP treatment (right). (C). The Spare respiratory capacity (SRC) of LSKs was calculated from the data shown in panel A (n=9 per genotype, from 3 independent experiments).

D, E. (D) Glycolysis Stress test. Extracellular acidification rates (ECAR) of LSKs were measured at basal conditions (unbuffered assay medium without glucose) and after sequential loading of glucose (7.5mM), Oligomycin (350nm), and 2-deoxyglucose (50mM; 2DG, a glucose analog). (E). AUC of baseline ECAR levels (left), and glycolytic capacity (maximal ECAR) was calculated from the data shown in panel D (n=7-8 per genotype, from 3 independent experiments).

F. Relative ATP content in indicated HSPC subsets determined using an ATP assay kit (n=6 mice per genotype).

G. Representative FACS histograms of MitoTracker Green fluorescence intensity on indicated HSPC subsets from control and cKO mice. Relative MitoTracker Green mean fluorescence intensity (MFI) is quantified below for n=6 mice of each genotype.

H. Representative electron microscopy images (4,800x magnification) of control and PKCδ cKO HSPCs. Arrows indicate the mitochondria enriched regions (n=3 mice per genotype). Scale bar represents 500nm.

I. ROS levels in control and PKCδ cKO HSPCs as measured by FACS based CM-H2DCFDA staining. Bar graph at right shows the relative CM-H2DCFDA MFI. Data compiled from three independent experiments (n=6-8 per genotype).

Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.
Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors (2018)
Figure 7

Figure 7.

PKCδ-deficiency impacts multiple signaling cascade and transcriptional network governing HSPC homeostasis and function.

A-C Quantitative RT-PCR analysis of selected (A) HSPC transcriptional regulators, (B) cell cycle and apoptosis regulators, and (C) known HSPC metabolic regulators expression in bone marrow HSPCs (n=4 mice per genotype).

D-M. Intracellular flow cytometry analysis of Cyclin D1 protein (D); pro-survival regulators Mcl1, Blc2, and Bim protein (E); representative FACS histograms and bar graphs show relative levels of pAKT (Ser473) relative to WT controls (F); pFoxO3a (Ser253) (G); Total cMyc protein (H); pRb (Ser807/811) (I); representative FACS histograms and bar graphs show total PPARγ protein levels relative to WT controls (J); Relative expression levels of total PPARδ protein in HSPCs (K); Activation of pmTOR (Ser2448) (L); pS6 (Ser235/236) and (Ser240/244) (M). Data shown in bar graphs in (D-M) are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target).

Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.

Expanded view figures
Bld10p/Cep135 determines the number of triplets in the centriole independently of the cartwheel (2022)
Figure 1

Figure 1. Expression of engineered Bld10ps and the extent of flagellation in transformed Chlamydomonas cells

(A) Schematic diagrams of the HA-tagged and truncated Bld10ps used in this study. Regions predicted to have high (>80%) and modest (>50%) probabilities of forming coiled-coils are shown in dark and light blue, respectively, and the positions of the 3×HA sequences are shown in red.

(B) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld10, bld10::B10-N-HA, bld10::B10-M-HA, and bld10::B10-C-HA cells using antibodies against the C-terminal region of Bld10p (Anti-Bld10-C antibody, upper blot) (Matsuura et al., 2004) and the HA antigen (Anti-HA antibody, lower blot). The apparent molecular weights of tagged Bld10ps are slightly larger than that of native Bld10p due to their HA tags.

(C) Percentages of flagellated cells in wild-type (CC-124), bld10, bld10::B10-N-HA, bld10::B10-M-HA, bld10::B10-C-HA, bld12, bld10bld12, bld10bld12::∆N3, bld10bld12::∆C2, bld10::∆N3, and bld10::∆C2 cells. Cells with two (red), one (green), and zero (blue) flagella were counted. n, number of cells counted.
Bld10p/Cep135 determines the number of triplets in the centriole independently of the cartwheel (2022)
Figure 2

Figure 2. Localization of Bld10p and the C-terminus of SAS-6 within the centriole

(A and B) Immunoelectron micrographs of centrioles in nucleo-flagellar apparatuses from bld10::B10-N-HA, bld10::B10-M-HA, bld10::B10-C-HA, and bld12::SAS-C-HA cells pretreated with an anti-HA antibody and a colloid gold-labeled secondary antibody. Longitudinal sections (A) and cross-sections (B) of the centrioles are shown.

(C) Localizations of gold particles plotted on a triangular map representing one-ninth of the cross-section of the proximal region of the centriole. The trapezoid area in the top panel is shown in the lower panels. The localizations of B10-N-HA, B10-M-HA, B10-C-HA, and SAS-C-HA are shown in red, yellow, green, and purple, respectively. Numbers of centriole images used for this analysis are 12, 7, 8, 8, respectively. n, numbers of gold particles. The coordinates of the four signals, as well as the signal of anti-Bld10p antibody obtained by a post-embedding method (blue, see below), are superposed in the lowermost panel.

(D) Immunoelectron micrographs of wild type centrioles treated with anti-Bld10p (B10-C) after embedding in the resin and sectioning. Localization of 16 gold particles in 13 centriole images are plotted on the triangular map (the right panel).

Data information: Scale bar, 100 nm.
Bld10p/Cep135 determines the number of triplets in the centriole independently of the cartwheel (2022)
Figure 4

Figure 4. Centriole structures in bld10bld12 cells expressing truncated Bld10ps in the absence of the cartwheel

(A) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld12, bld10, bld10bld12, and bld10bld12::∆N3 cells using an antibody against the C-terminal one-third of Bld10p (Anti-Bld10-C antibody). The band corresponding to truncated Bld10p (Bld10p ∆N3) overlaps with a non-specific band at 75 kDa.

(B) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld12, bld10, bld10bld12, and bld10bld12::∆C2 cells using an antibody raised against the N-terminal 120 amino-acid sequence of Bld10p (Anti-Bld10-N antibody).

(C) Cross-sectional electron micrographs of centrioles in bld12, bld10::∆N3, bld10bld12::∆N3, bld10::∆C2, and bld10bld12::∆C2 cells. Scale bar, 100 nm.

(D) Distribution of the number of triplets in centrioles of bld12, bld10::∆N3, bld10bld12::∆N3, bld10::∆C2, and bld10bld12::∆C2 cells. Red lines denote the normal number of microtubules in the centriole (nine). n, number of centrioles observed.