EMBO/soda-nel huggingface dataset:
Caldendrin and myosinV regulate synaptic spine apparatus localization via ER stabilization in dendritic spines (2021)
Figure 4

Figure 4: The presence of caldendrin does not impair the ability of myosin V to bind actin filaments in vitro and in live COS7 cells.

A Commasie blue stained SDS-PAGE showing results of F-actin co-sedimentation assay. In vitro polymerized actin filaments sediment irrespectively of the presence and absence of calcium ions. BSA was used as a control to estimate the amount of "passively" co-sedimented non-actin-binding protein and/or residual non-actin-binding protein in the pellet. There was no difference between the amount of BSA in the pellet with or without F-actin.

B Co-sedimentation assay of GFP-tagged myosin fragment lacking the globular tail domain (GFP-myoVa-motor+CC) with F-actin shows that the presence of Ca2+-caldendrin does not prevent myoV from binding to actin filaments. GFP-myoVa-motor+CC, as visualized by an anti-GFP antibody via western blot, co-sediments with F-actin (comassie blue stained SDS-PAGE), both in presence and absence of Ca2+, regardless of the presence of caldendrin.

C Schematic representation of the inducible transport assay in COS7 cells. Three plasmids are co-expressed in COS7 cells: a constitutively active kinesin-2 motor (KIF17) fused to a GFP-tagged peroxisome targeting sequence (KIF17-GFP-PEX13), an RFP-tagged PEX13 fused to an FKBP protein (PEX-RFP-FKBP), and a constitutively active myosin Vb motor fused to an FRB protein (myoV-GFP-FRB). Addition of the small molecule rapalog leads to dimerization of the FKBP and FRB, thus recruiting myoV to the peroxisome. Additionally, the cells were transfected with untagged caldendrin (or an empty control vector) to assess the effect of caldendrin binding to myoV on its activity in living cells.

D Left: Representative image of COS7 cell expressing the inducible dimerization system described in C. Red: Peroxisomes (PEX-RFP-FKBP), Green: KIF17-GFP-PEX13 and myoV-GFP-FRB. Scale bar = 10 µm. Middle and right: Individual tracks of kinesin-driven peroxisome movements reconstructed over 20 seconds (yellow) before (middle) and after (right) addition of rapalog indicate that the recruitment of myoV slows down the motility of peroxisomes. Blue outlines represent manually drawn cellular border and nucleus (excluded from analysis). Also see Movie EV2.

E Analysis of the mean squared displacement (MSD) of peroxisomes over time (left) suggests a linear increase for all conditions. Slope analysis for MSD curves for individual cells (right) indicates that addition of rapalog induces efficient stalling (2-way ANOVA, *** p<0.0001 as compared before and after rapalog). However, no difference between control and caldendrin (cald) transfected cells could be observed (2-way ANOVA, Mean ± SEM; n control=14 cells, n cald=23 cells; 3 experiments).
Caldendrin and myosinV regulate synaptic spine apparatus localization via ER stabilization in dendritic spines (2021)
Figure 5

Figure 5: Overexpression of caldendrin leads to an increase of stable SER and the spine apparatus in dendritic spines of rat primary hippocampal neurons.

A Example confocal images from 10 min time-lapse imaging of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons (DIV17) expressing an ER-marker (ER-tDimer). Yellow arrows show dendritic protrusions with a stably inserted ER (>10 min). Blue arrows indicate protrusions with a transient ER entry. Scale bar = 5 µm. Also see Movie EV4.

B Quantification of dynamic SER presence in protrusions of control (GFP) or caldendrin (cald-GFP) transfected neurons. In protrusions of cald-GFP neurons, the percentage of stably localized ER (> 10 min) is increased, while transient SER visits are decreased compared to the control. (mean ± SEM, 2-way ANOVA, * p=0.0386 for interaction, n GFP = 9, n cald-GFP = 10; 2 experiments)

C Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons expressing an ER-marker (ER-tDimer). To pre-load overexpressed caldendrin with Ca2+, neurons were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation and stained with a homer1-antibody to visualize synapse-containing spines. Arrows indicate spines that contain ER. Scale bar = 5 µm.

D - F Quantification of protrusion- and spine density and the number of SER-positive spines as shown in C. (mean ± SEM, n GFP ctrl = 24 cells, n Cald-GFP = 25 cells; 3 experiments). (D) The density of dendritic protrusions (spines and filopodia) is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0119, unpaired t-test). (E) The density of homer1-positive dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (** p=0.0049, unpaired t-test). (F) The percentage of ER-containing dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0179, unpaired t-test)

G Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons pre-treated with bicuculline as in C and stained against homer1 and the spine apparatus-marker synaptopodin. Yellow arrows indicate synaptopodin clusters localized inside the dendritic shaft, blue arrows indicate spine-localized synaptopodin. Scale bar = 5 µm.

H - J Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in G. (mean ± SEM, n GFP ctrl = 21 cells, n cald-GFP = 30 cells; 2 experiments). (H) Spine density is increased in cald-GFP overexpressing neurons compared to the control (in line with 5e). (** p = 0.0024, unpaired t-test). (I) The total number of synaptopodin clusters is not affected by caldendrin overexpression. (n.s., unpaired t-test). (J) The percentage of synapotpodin-positive spines is increased in cald-GFP overexpressing neurons compared to the control. (**** p < 0.0001, unpaired t-test).
Caldendrin and myosinV regulate synaptic spine apparatus localization via ER stabilization in dendritic spines (2021)
Figure 6

Figure 6: Loss of caldendrin leads to an increased frequency of SER entry and decreased fraction of stable SER in dendritic protrusions of mouse primary hippocampal neurons.

A 10 min time-lapse imaging of DIV12 wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO + cald-GFP) or caldendrin knock-out treated with jasplakinolide (cald KO + JPK) mouse primary hippocampal neurons, additionally expressing a cell fill (maxGFP) and an ER-marker (ER-tDimer). Yellow arrows indicate protrusions with a stably anchored ER (>10 min). Blue arrows indicate protrusions that show transient presence of ER. Scale bar = 5 µm.

B Quantification of dynamic SER presence in protrusions of cald WT, cald KO, cald KO + cald-GFP and cald KO + JPK neurons across individual cells, suggesting higher dynamics for cald KO and cald KO + JPK neurons as seen from an increase of protrusions with transiently present ER compared to cald WT neurons, whereas the opposite can be seen for cald KO + cald-GFP. (mean ± SEM, 2-way ANOVA, ***p<0.001 for overall interaction, Bonferroni post-tests: *p<0.05 and **p<0.01 for cald WT vs cald KO + cald-GFP, for absent and permanent protrusions, respectively; ***p<0.001 for cald WT vs cald KO + JPK for absent protrusions; ***p<0.001 for cald KO vs cald KO + cald for permanent protrusions; **p<0.01 for cald KO + cald-GFP vs cald KO + JPK for both absent and permanent protrusions. cald wt, n=13 cells, 3 experiments; cald KO, n=23 cells, 3 experiments; cald KO + cald-GFP, n=11 cells, 2 experiments; cald KO + JPK, n=8 cells, 2 experiments)

C Further analysis of the data in (B) for all transiently visited protrusions. In cald WT neurons, roughly 50% of protrusions get visited by an ER only once, while the other half experiences multiple ER entries. This ratio is changed towards multiple ER entries in cald KO neurons, and can be rescued by overexpression of caldendrin-GFP (cald KO + cald), but not by 48 h treatment with 50 nM jasplakinolide (cald KO + JPK). The n of independent experiments, cells and analysed protrusions is the same as in (B).
Caldendrin and myosinV regulate synaptic spine apparatus localization via ER stabilization in dendritic spines (2021)
Figure 7

Figure 7: Loss of caldendrin decreases the number of synaptopodin-positive spines, which can be rescued by caldendrin overexpression, but not by the F-actin stabilization

A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin. The groups compared are wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO cald), and caldendrin knock-out treated with jasplakinolide (cald KO JPK). Scale bar = 5 µm.

B - C Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in A (mean ± SEM. Kruskal-Wallis test with Dunn's multiple comparisons post-test. *** p < 0.0001. cald wt n = 34 cells, 2 experiments; cald KO n = 33 cells, 3 experiments; cald KO cald n = 10 cells, 1 experiment; cald KO JPK n = 14 cells, 1 experiment). (B) The total number of synaptopodin clusters is unaffected by the loss of caldendrin. (C) The number of synaptopodin-positive spines is reduced in cald KO compared to the WT neurons. This phenotype could be rescued by over-expression of caldendrin (cald KO + cald), but not by treatment with JPK (cald KO + JPK).

D Model depicting the suggested mechanism of spine apparatus formation. Increased activity results in increased ER visits to the spine, which is mediated by calmodulin-associated myoV. In a subset of spines that are enriched with caldendrin, calcium-dependent myoV-caldendrin interaction transforms the processive motor into an F-actin tether, thereby stabilizing the ER in the spine. Subsequent accumulation of synaptopodin results in the formation of the spine apparatus.

Expanded View Figure legends
Ketone body oxidation increases cardiac endothelial cell proliferation (2022)
Figure 1

Figure 1. Key enzymes of ketone body oxidation are expressed in cardiac endothelial cells.

(A) Oxidation of ketone bodies requires the enzymes SCOT and BDH1 and yields acetyl-CoA which enters the tricarboxylic acid (TCA) cycle. (B) Immunoblot of SCOT, BDH1 and the loading control VCP across various organs obtained from adult C57Bl/6J mice. (C, D) Quantification of SCOT and BDH1 expression levels relative to VCP in various murine organs. (E) Immunoblot of SCOT, BDH1, CD31 and the loading control VCP in murine endothelial cells isolated from heart, muscle, lung and adipose tissue. (F) Immunoblot of SCOT, BDH1, CD31 and the loading control VCP in human cardiac ECs, HUVECs and adipose tissue ECs. (G) Immunoblot of SCOT, BDH1, CD31 and VCP in cardiac endothelial cells (EC) and non-endothelial cell fraction (non-EC) isolated from adult C57Bl/6J mice. (H) Immunoblot of SCOT, BDH1 and β-actin in immortalized murine cardiac endothelial cells (MCEC). (I, J) Ketone body concentration in cell culture medium of MCEC treated with 2 mM D-β-hydroxybutyrate (β-OHB) or 2 mM acetoacetate (AcAc) after 24 hours. Data are presented as mean ± SD. n=3; *, p<0.05; ***, p<0.001 unpaired Student's t-test.
Ketone body oxidation increases cardiac endothelial cell proliferation (2022)
Figure 4

Figure 4. Ketone body treatment does not increase sprouting capacity in SCOT-deficient cardiac endothelial cells.

(A) SCOT-deficient murine cardiac endothelial cells were generated using the CRISPR/Cas9 technology by targeting the SCOT-encoding gene Oxct1. A non-targeting control construct was used to generate the non-targeting control cells (nt ctrl). Immunoblot of SCOT and β-actin in non-targeting control cells (nt ctrl) and Oxct1 knockout cells (Oxct1 ko). (B, C) Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. The average number of sprouts per spheroid and the accumulated sprout length was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test.
Ketone body oxidation increases cardiac endothelial cell proliferation (2022)
Figure 6

Figure 6. Ketogenic diet induces proliferation of cardiac endothelial cells in mice.

(A, B) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU, double positive cells are indicated by arrowheads, and quantification of double positive cells in heart sections per high power field (HPF). (C, D) Quantification of Ki67+/ERG+ cells in heart sections of mice kept on a ketogenic diet or control diet for four weeks or six weeks. (E) Quantification of CD31+ area in heart sections of mice kept on a ketogenic diet or control diet for four months. (F) Quantification of Ki67+ endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks (gastrocnemius and soleus muscle) or two weeks (brain, lungs, subcutaneous adipose tissue, liver). Scale bar: 50 μm. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.
Ketone body oxidation increases cardiac endothelial cell proliferation (2022)
Figure 7

Figure 7. Ketogenic diet prevents vascular rarefaction in mice after transverse aortic constriction.

(A) Parameters of cardiac function (fractional shortening, left ventricular end-diastolic anterior wall thickness (LVAW;d), ratio of left ventricular (LV) mass to body mass) eight weeks after transverse aortic constriction (TAC) in male and female C57Bl/6J mice receiving a control diet or ketogenic diet compared to sham animals kept on the same diet. (B) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG. (C) Quantification of Ki67+/ERG+ cells per high power field (HPF) in heart sections of mice kept on a control diet or a ketogenic diet eight weeks after TAC surgery. (D) Quantification of isolectin IB4-positive vessels per high power field (HPF) in heart sections of mice kept on a control diet or a ketogenic diet eight weeks after TAC surgery relative to sham animals from the respective diet group. Scale bar: 50 µm. Data are presented as mean ± SD. n≥4; *, p<0.05; ***, p<0.001 unpaired Student's t-test.
Transfer of LncRNA CRNDE in TAM-derived exosomes is linked with cisplatin resistance in gastric cancer (2021)
Figure 1

Figure 1 LncRNA CRNDE is enriched in tumor-associated macrophages of gastric cancer patients.

(A) LncRNA CRNDE expression level was measured by qRT-PCR in cancerous tissues and adjacent normal tissues obtained from 35 cases of gastric cancer (GC) patients.

(B) The correlation between LncRNA CRNDE expression and the proportion of CD63+CD163+ M2 macrophages in GC cancerous tissues (n = 7) was determined by the Pearson correlation coefficient.

(C) LncRNA CRNDE expression level was measured by qRT-PCR in adjacent normal tissue-derived macrophages (NTMs) and tumor-associated macrophages (TAMs) obtained from 7 cases of GC patients.

Data information: Data are expressed as mean ± SD.  A and C: Student's t-test; B: Spearman's correlation analysis. **P<0.01.
Transfer of LncRNA CRNDE in TAM-derived exosomes is linked with cisplatin resistance in gastric cancer (2021)
Figure 2

Figure 2 LncRNA CRNDE is enriched in M2-polarized macrophages and in M2 polarized macrophage-derived exosomes (M2-exo).

(A, B) Mouse bone marrow-derived macrophages (BMDMs) and human peripheral blood monocytes were induced towards an M2-polarized phenotype by the stimulation of 20 ng/ml interleukin 4 (IL-4) plus 20 ng/ml IL-13. LncRNA CRNDE expression level in (A) BMDMs, BMDM-polarized M2 macrophages, (B) monocytes, and monocyte-polarized M2 macrophages was detected by qRT-PCR.

(C) and (D) Left: representative electron micrograph of exosomes isolated from the medium of BMDMs, BMDM-polarized M2 macrophages, monocytes and monocyte-polarized M2 macrophages are depicted, showing the typical morphology of exosomes. Upper right: the protein levels of CD63 and CD81 were measured by Western blot. Lower left: Nanoparticle tracking analysis showed that most of the particles had a similar size of 50-150 nm in diameter with a peak at around 100 nm. Lower right: LncRNA CRNDE expression levels in different exosomes.

Data information: Scale bar= 100 nm. The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. **P<0.01 vs. BMDMs or monocytes or BMDMs-exo or monocytes-exo.
Transfer of LncRNA CRNDE in TAM-derived exosomes is linked with cisplatin resistance in gastric cancer (2021)
Figure 4

Figure 4 LncRNA CRNDE induces cisplatin resistance in GC cells.

(A-D) Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). Then, MFC cells were treated with indicated exosomes and cisplatin (CDDP; 1μg/ml) for 48 h. (A) LncRNA CRNDE expression in indicated exosomes was detected by qRT-PCR. (B) The survival rate of MFC cells was measured by cell counting kit-8 (CCK-8) assay. (C) Representative images of colony formation assay performed on MFC cells. (D) Cell apoptosis was measured by flow cytometry.

(E-H) Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). Then, SGC7901 cells were treated with indicated exosomes and CDDP (1μg/ml). (E) LncRNA CRNDE expression, (F) cell survival rate, (G) cell proliferation, and (H) cell apoptosis were measured.

Data information: (A, D, E, and H): The results shown are from three biological replicates. (B and F): The results shown are from seven biological replicates. Data are expressed as mean ± SD. (A, B, D, E, F, and H): one-way ANOVA. **P<0.01.
Transfer of LncRNA CRNDE in TAM-derived exosomes is linked with cisplatin resistance in gastric cancer (2021)
Figure 5

Figure 5 LncRNA CRNDE induces CDDP resistance in vivo.

3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected.

(A) Growth curves of MFC subcutaneous homograft tumors (The homograft tumor sizes were recorded from the day of CDDP treatment). *P<0.05, **P<0.01 vs. BMDMs-exo; #P<0.05, ##P<0.01 vs. M2-si-control-exo.

(B, C) The homograft tumors (B) photos and (C) weight of each mouse on day 6 after CDDP injection. **P<0.01.

(D) LncRNA CRNDE expression in homograft tumors of mice. **P<0.01.

Data information: Data are expressed as mean ± SD. (A, C and D): one-way ANOVA.
Transfer of LncRNA CRNDE in TAM-derived exosomes is linked with cisplatin resistance in gastric cancer (2021)
Figure 6

Figure 6 LncRNA CRNDE negatively regulated PTEN expression

(A) RNA Immunoprecipitation (RIP) and (B) RNA pull-down assays were conducted to detect the association of LncRNA CRNDE and phosphatase and tensin homolog (PTEN)/ neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1).

(C) SGC7901 cells were transfected with pcDNA-CRNDE or its negative control (Mock) before MG132 treatment (10 µM, 6 h). The Co-immunoprecipitation (Co-IP) assay was performed to examine the combination of PTEN and NEDD4-1.

(D) The expressions of PTEN and NEDD4-1 were measured using western blot in SGC7901 cells transfected with pcDNA-CRNDE or Mock. GAPDH was served as an internal control.

(E) The expressions of PTEN and NEDD4-1 were measured in SGC7901 cells transfected with pcDNA-CRNDE or Mock at four time-points after cycloheximide (CHX, 100 μg/mL) treatment. GAPDH was served as an internal control. Left: Representative bands. Right: Quantitative results. The results shown are from three biological replicates and are expressed as mean ± SD. Student's t-test. *P<0.05, **P<0.01 vs. Mock.

(F, G) The ubiquitylation of PTEN was measured in (F) SGC7901 cells transfected with pcDNA-CRNDE or Mock, and (G) 293T cells transfected with indicated vectors.
Transfer of LncRNA CRNDE in TAM-derived exosomes is linked with cisplatin resistance in gastric cancer (2021)
Figure 7

Figure 7 PTEN mediated the modulatory effect of exosomal LncRNA CRNDE on the sensitivity of GC cells to CDDP.

(A-D) Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. (A) The protein levels of PTEN, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), p-glycoprotein (p-gp), B-cell lymphoma 2 (Bcl-2) were measured. GAPDH was served as an internal control. The (B) survival rate, (C) cell proliferation, (D) cell apoptosis in SGC7901 cells.

(E-G) SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell survival rate, cell proliferation, and cell apoptosis were measured.

Data information: (B and E): The results shown are from seven biological replicates. (D and G): The results shown are from three biological replicates. Data are expressed as mean ± SD. (B, D, E, and G): one-way ANOVA. **P<0.01.

Expanded View Figure legends
Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes (2021)
Figure 1

Figure 1. Establishing and characterizing isogenic female/male ESCs

A Schematic representation showing how the ESCs were established.

B Sexing the established ESCs by detecting the Y-linked Zfy gene using genomic DNA (gDNA)-qPCR, normalized to Gapdh. n.d.: not detected. Average of presumed males was set to 1.

C DNA-FISH detection of the number of X-linked Atrx gene foci. Representative images of the DNA-FISH are shown. Nuclei are surrounded by white dotted lines. Magenta: Atrx (white arrowheads), cyan: DAPI. N>100.

D PCA from RNA-seq.

E Heatmap analysis from RNA-seq. Each log10(FPKM+1) value was used for clustering. Genes with high or low expression are shown in red and blue, respectively. FPKM: fragments per kilobases per million reads.

F X and Y chromosome mapping. Genes with high expression (up) are shown in orange and low expression (down) are shown in blue. The defined ratio of up or down genes is > 1.5 and < two-thirds, respectively. The comparison is (each female vs the average for males) or (each male vs the average for females). The location controls for the expressed genes (exp. genes) on the X-chromosome and Y-chromosome are plotted (red squares) for genes whose FPKM averages were above 1 for females or males.

Data information: Data in (B) are derived from two technical replicates and are shown as the mean with single data points of both experiments. Scale bar in (C) is 10 μm. N represents number of cells.