EMBO/soda-nel huggingface dataset:
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and -independent mechanisms (2021)
Figure 3

Figure 3: dTor affects the fat body lipidome via Nep1r1, Ctdnep1 and Lipin

A) dTorKO larvae expressing UAS RNAi transgenes or expressing a wild-type dTor cDNA (positive control). The fat body is pseudo-colored red.

B) Topology of NEP1R1, CTDNEP1, and Lipin.

C) Fat body cells labeled by BODIPY to detect neutral lipids.

D) The relative abundance of each lipid class (mean ± SEM) within the set of six membrane lipids. n = 3 MS analyses of 4DO fat body samples for each genotype. ‡ symbols indicate a significant difference compared to w- expressing Luciferase RNAi, * symbols indicate a significant difference compared to dTorKO expressing luciferase RNAi. Note we only tested the effect of Lipin, Ctdnep1, and Nep1r1 RNAi when a lipid class significantly differed between w- and dTorKO. One-way ANOVA, Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ‡‡‡‡ p < 0.0001.

E) As above except that TAG abundance was calculated relative to protein. One-way ANOVA, Tukey's multiple comparisons test.

F, G) Bars show the fraction (mean ± SEM) of membrane GL and GPL lipids with each fatty acyl chain characteristic. n = 3 MS analyses of 4DO fat body samples for each genotype. MUFA, mono-unsaturated; PUFA, poly-unsaturated. ‡ indicate a significant difference between w- and dTorKO expressing Luciferase RNAi, * indicate a significant difference compared to dTorKO expressing Luciferase RNAi. Two-way ANOVA, Dunnett's multiple comparison test. ‡ p < 0.05 and * p < 0.05.
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and -independent mechanisms (2021)
Figure 4

Figure 4: CTDNEP1 and Lipin concentrate at the NE of fly and mouse cells lacking Torsins

A - D) Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg-Gal4 driver, and labeled with anti-Calnexin (red) and a neutral lipid dye (LipidTOX, magenta). Note that dTorKO cells are smaller. White arrowhead indicates NE enrichment of LipinmGFP.

E, F) CTDNEP1Scarlet and anti-Sun1 localization in MEFs.

G, H) Lipin1mGFP localization in HEK293T cells transfected with a plasmid that co-expresses CTDNEP1myc-WT or -CD (red) and NEP1R1V5 (magenta). (G') Upper: enlargement of panel G, white arrow highlights NE enrichment. Lower: plot showing the intensity of Lipin1mGFP signal (mean ± SD, n = 5 cells) along a 3 µm profile that transects the NE at 0 µm.

I - K) Mouse Lipin1mGFP expressed in MEFs, red signal shows Calreticulin.

L) Percentage of MEFs with primarily cytosolic or nuclear Lipin1mGFP. Chi-square test. **** p < 0.0001.
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and -independent mechanisms (2021)
Figure 5

Figure 5: dTor loss impairs NPC insertion during nuclear growth in developing fat body cells

A) Interphase NPC insertion occurs via inside-out extrusion of the INM, followed by INM/ONM fission and fusion (Otsuka & Ellenberg, 2018). Nucleoporins are not depicted.

B) The mature NPC, highlighting proteins examined in fat body cells. Green, cytosolic filaments; yellow, outer ring subcomplex; pale blue, inner ring subcomplex; brown, transmembrane nucleoporins; red, phenylalanine-glycine repeats (FG-Nups) that form the permeability barrier; white/blue, nuclear basket Nups.

C) Fat body nuclei increase in size during development. Graph shows mean ± SD for each day during development of larvae. DO, day-old.

D - M) Control (w-) and dTorKO fat body nuclei labeled with anti-LaminDm0, Megator/Tpr, mAb414, or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg-Gal4 driver.

N, O) Representative TEM of the NE of control and dTorKO 5DO fat body cells. Red arrows show nuclear pores. Nuc, nucleus; Cyto, cytosol.

P) The percentage of NE in individual 5DO fat body cells that has normal INM, ONM, and NPC morphology. Black lines show the mean of each group.

Q, R) Representative TEM showing the NE of 5DO dLap1-/- fat body cells. Red arrows in (Q) show nuclear pores. Dashed yellow line highlights unusual circular membrane profiles.
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and -independent mechanisms (2021)
Figure 6

Figure 6: Nep1r1 and Ctdnep1 downregulation restore NPC levels and suppress INM herniations in dTorKO cells

A) mAb414 labeling of 5DO dTorKO fat body cells expressing the dTor cDNA, Luciferase RNAi, or RNAi against Nep1r1, Ctdnep1, or Lipin. Nuc, nucleus.

B) The percentage of fat body cells with NE-localized mAb414. ** and **** indicate that Lipin, Ctdnep1, and Nep1r1 RNAi significantly increased the number of dTorKO cells with NE-specific mAb414 labeling compared to the Luciferase RNAi (Luc). Chi-square test followed by individual post hoc Chi-square tests. ** p < 0.01 and **** p < 0.0001.

C) NE ultrastructure in 5DO dTorKO cells. Red arrows, nuclear pores; yellow arrows, abnormal INM herniations. Nuc, nucleus; Cyto, cytosol.

D) The density of NPC detected by TEM in the 5DO dTorKO NE. Each point shows the mean of multiple measurements from an individual nucleus, bars show mean ± SD for the group (n = 5). One-way ANOVA, with Bonferroni's post hoc test. ** p < 0.01, *** p < 0.001 and **** p <0.0001.

E) Same as above except that the density of INM herniations is presented.

F) The percentage of NE in 5DO fat body cells that has normally appearing INM, ONM, and NPC morphology. Points show values from individual nuclei (n = 5). Bars show mean ± SD. Grey zone shows +/- 2x SD of the positive control mean, dotted red line shows mean of dTorKO expressing Luciferase RNAi. One-way ANOVA, with Bonferroni's post hoc test. ** p < 0.01 and **** p <0.0001.

G) The percentage that Lipin, Ctdnep1, and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body. 0% "normal" reflects lipid abundance in dTorKO expressing Luciferase RNAi, while 100% "normal" reflects lipid abundance in w- expressing Luciferase RNAi. Percentage normalization was separately calculated for PI, PA, DAG, PC, PE and PS (for dTorKO expressing Lipin, Ctdnep1, and Nep1r1 RNAi). Points show the mean of all lipid class normalization per sample, bars show mean ± SEM for the group.
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and -independent mechanisms (2021)
Figure 7

Figure 7: Abnormal nucleocytoplasmic membrane channels occur in fat body cells overexpressing NEP1R1, CTDNEP1 or Lipin, and dLap1-/- cells

A - C) Fat body cells expressing GFP tagged UAS transgenes with r4-Gal4.

D) TAG (nmol/ μg protein) in the 4 DO fat body expressing UAS transgenes with the r4-Gal4 driver. Bars show group mean +/- SEM (n = 6), points show values from individual MS runs. Unpaired T-Test. ** p < 0.01.

E - I) NE ultrastructure of 5DO fat body cells overexpressing UAS transgenes, or from dLap1-/- animals. Yellow dotted lines highlight NE regions with subtly abnormal membrane architecture. Red arrows indicate nuclear pores. R1, region 1; R2, region2; Nuc, nucleus; Cyto, cytosol.

J - M) Data from FIB-SEM imaging of fat body nuclei expressing Sec61b (control), or CTDNEP1mGFP. (J - L) 3D reconstructions, (M) distance between the INM and ONM at NPC in wild-type cells versus CTDNEP1mGFP-induced structures. Kolmogorov-Smirnov non-parametric T-Test. **** p < 0.0001.

N, O) TEM analysis of the NE in 5DO fat body cells expressing Ctdnep1 with DAGK or Sec61 (control). Red arrows indicate nuclear pores.
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and -independent mechanisms (2021)
Figure 8

Figure 8: Impaired Nup35 recruitment can explain the abnormal membrane channels caused by excess PA to DAG conversion

A) The NPC is comprised of several subcomplexes.

B - G) Confocal imaging of 5DO fat body nuclei expressing UAS transgenes and labeled by anti-Megator/Tpr, Nup214, Nup35, or mAb414, or expressing Nup107RFP.

H) mAb414 localization in 5DO fat body cells expressing the indicated UAS transgenes or from a dLap1-/- animal. *** and **** indicate significant difference compared to control mGFPSec61b expressing cells. Chi-square test followed by individual post hoc Chi-square tests. *** p < 0.001 and **** p < 0.0001.

I - K) NE ultrastructure of 5DO fat body cells from a (I) control animal, (J) homozygous Nup35BG01311 and (K) homozygous Nup35MB02683 animals. Nuc, nucleus; Cyto, cytosol.

L) Model of the relationship between nucleocytoplasmic channels, PA metabolism, and NPC maturation.
Programmable CRISPR-Cas transcriptional activation in bacteria (2020)
Figure 1

Figure 1. A high-throughput platform to identify and engineer bacterial CRISPR-Cas transcription activators (CasTAs).

(A) General strategy for bacterial CRISPRa using a dCas9 fused with a transcriptional activator, a targeting gRNA, and reporter genes.

(B) System components are constructed in three compatible plasmids. CasTA candidates could be cross validated through GFP and antibiotic resistance gene reporters.

(C) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA. The dCas9-ω was verified in the ∆rpoZ strain background, and the rest of candidates were verified in the wild-type strain (BW25113).

(D) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9-AsiA with gRNA-H3 under kanamycin selection (2.5 μg/ml).

(E) Different gRNAs were paired with dCas9-AsiA and dCas9-ω to profile the optimal gRNA binding distance. The dCas9-AsiA was verified in the wild-type strain (BW25113), and dCas9-ω was examined in the ∆rpoZ strain background.

(F) Comparing single and multiple gRNA with dCas9-AsiA.

Data information: Data in all panels are 3-5 biological replicates with +/- standard error of mean (SEM).
Programmable CRISPR-Cas transcriptional activation in bacteria (2020)
Figure 2

Figure 2. Directed evolution of dCas9-AsiA for higher potency.

(A) Schematics of two rounds of directed evolution to improve potency of dCas9-AsiA. Pie charts show frequencies of dCas9-AsiA variants identified from each round.

(B) Mutations found in enriched AsiA variants and their positions along the AsiA secondary structure (left). Crystal structure of wild-type AsiA (blue) interfaced with region 4 of σ70 (orange) are shown (right).

(C) Distribution of fluorescence signal of the GFP reporter induced by different dCas9-AsiA variants (top). Fold induction by different dCas9-AsiA variants is shown (bottom).

(D) CRISPRa induction of promoters with varying basal expression levels. CRISPRa-, basal expression of the promoter; CRISPRa+, expression activated by dCas9-AsiA-m2.1 and associated gRNA-H4.

Data information: Data shown are 3 biological replicates with +/- SEM.
Programmable CRISPR-Cas transcriptional activation in bacteria (2020)
Figure 3

Figure 3. Evolved CasTA2.1 could activate genomic targets and mediate multiplexed gene activation and repression.

(A) CasTA2.1 upregulates a genomically inserted GFP reporter.

(B) Chromosomal gene targets activated by CasTA2.1 with bars showing the activation fold change and dots showing basal expression of each gene. Expression was quantified using RT-qPCR.

(C) CasTA2.1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene. A non-specific gRNA is used as the negative control.

(D) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2.1 on a reporter containing GFP and mScarlet. Parental cells had low basal GFP and high basal mScarlet expression.

Data information: Data shown are 3-4 biological replicates with +/- SEM.
Programmable CRISPR-Cas transcriptional activation in bacteria (2020)
Figure 5

Figure 5. Evolved CasTA functions in multiple bacterial species.

(A) Multiple sequence alignment of AsiA homologs from different phage genomes at residue positions 50-61. Highlighted red residues indicate positions that are mutated in AsiA-m2.1.

(B) CRISPRi in S. enterica and K. oxytoca using CasTA2.1.

(C) CRISPRa in S. enterica and K. oxytoca using CasTA1.0 (dCas9-AsiA_wt), CasTA2.1 (dCas9-AsiA_m2.1) or ancestral strain with basal promoter expression (none).

Data information: All data are 3-4 biological replicates with +/- SEM. * Student's t-test, p<0.0001 NS = non-significant.
Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis (2020)
Figure 1

Figure 1. Enrichment of lamin B1 in ANSPCs.

A. Enrichment of lamin B1 in the SGZ of DG.

B. Immunohistochemical analyses of lamin B1-high cells. The lamin B1-high cells express Sox2 or PSA-NCAM, but not Prox1. Arrowheads indicate Prox1 negative lamin B1-high cells in the SGZ.

C. Immunohistochemical analyses of lamin B1 in pNestin-GFP mice. An open arrowhead indicates RGL-ANSC and a closed arrowhead indicates a non-RGL(NR)-NPC.

D. A schema for the changes in lamin B1 levels during adult hippocampal neurogenesis.

E. Immunocytochemistry of lamin B1 in NPCs and Tuj1+ differentiating neurons 4 days after the induction of differentiation.

F. Relative expression of Lmnb1 and Tubb3 revealed by qRT-PCR from NPCs and differentiating neurons at the indicated days. ***P < 0.001, **P < 0.01, *P < 0.05, one sample t-test (n = 4).

G. Quantification of signal intensity of lamin B1 immunoreactivity in different neural cell types. F (3,82) = 173.1, P = 1.2 e-34, n = 12-30 cells per cell type, one-way ANOVA followed by Tukey-Kramer test (**P < 0.01).

Data information: A.U. = Arbitrary units. Bar graphs show mean ± s.e.m. Scale bars, 75 µm in (A), 25 µm in (B lower, C), 10 µm in (B upper, E).
Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis (2020)
Figure 2

Figure 2. Age-dependent reduction in lamin B1 in ANSPCs.

A. Age-dependent reduction of lamin B1 in the SGZ. As early as 5.5 months of age, the levels of lamin B1 in the SGZ were markedly reduced. Insets show higher magnification images in the SGZ.

B-D. Quantification of lamin B1 levels using immunofluorescent signals. Lamin B1 levels in Sox2-positive, PSA-NCAM-negative-ANSPCs were selectively downregulated at 5.5 months of age (ANPCs, P = 1.02129e-19; Neuroblasts, P = 0.09, DGCs, P = 6.4011e-15, ANOVA). ** P < 0.001, ANOVA followed by Tukey-Kramer test. (3 animals, 30 cells per conditions)

E. Age-dependent reduction of lamin B1 in RGL-ANSCs (upper) and NR-ANPC (lower) from 2 to 5.5 months of age. Arrowheads indicate RGL-ANSCs or NR-ANPCs.

F. Quantification of lamin B1 levels in RGL-ANSCs and ANPCs. * P < 0.05, *** P < 0.001, (4 animals, 18-32 cells per conditions, Mann-Whitney test).

G. A hypothetical model. Aging induces the reduction of lamin B1 in ANSPCs, which underlies stem cell aging.

Data information: Scale bars, 100 µm in (A), 10 µm in (E). Bar graphs show mean ± s.e.m. A.U. = Arbitrary units.
Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis (2020)
Figure 3

Figure 3. Precocious reduction of lamin B1 in ANSPCs induces anxiety-like behaviors.

Schematic of TAM treatment and behavioral tests.

B-F. Open field (OF) test. (B) Color-coded distribution of the exploratory path in the open field test. X-Y axes of squares correspond to the OF arena. Old WT mice as well as Lmnb1-cKO mice spend more time in the periphery of the OF arena. (C) Total distance traveled during the OF test. No significant difference among groups. F(2, 25) = 2.23, P = 0.13, ANOVA. (D) Velocity of travel during the OF test. F(2, 25) = 1.60, P = 0.22, ANOVA. N.S. (not significant). (E) Fraction of time spent in the periphery. F(2, 25) = 10.71, P = 0.00044, ANOVA followed by Tukey Kramer, ** P < 0.01. (F) Fraction of traveled distance in the periphery, F(2, 25) = 3.84, P = 0.035, ANOVA followed by Tukey Kramer, * P < 0.05 (n: WT = 10, WT-old =7, Lmnb1-cKO = 11 for the OF test).

G. Principal components analysis of behavior from the OF test (n: WT = 10, WT-old =7, Lmnb1-cKO = 11 for the OF test). Circles indicate 66% confidence levels for each group.

H-J. Novelty suppressed feeding. (H) Total feeding time during the NSF test, P = 0.0068, Kruskal-Wallis test followed by Dunn's test. ** P < 0.01. (n: WT = 6, WT-old =7, Lmnb1-cKO = 6 for novelty suppressed feeding). (I) Food consumption during the NSF test, P = 0.014, Kruskal-Wallis test followed by Dunn's test. * P < 0.05. (J) Total number of feeding instances during NSF test. N.S. (not significant).

Data information: Bar graphs show mean ± s.e.m.
Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis (2020)
Figure 4

Figure 4. Lamin B1-knockout promotes differentiation of ANPCs in the short term.

A. Schematic of TAM treatment and collection of brain tissue.

B, C. Representative images of Control and cKO with EYFP and DCX staining three weeks after TAM treatment. Note that the numbers of DCX-positive cells are increased. Boxes indicate magnified regions in C.

D, E, F. Quantification three weeks after TAM treatment. Among EYFP-positive cells, the density of RGL-ANSCs is not different (P = 0.65, t-test) (D). The density of ANPCs is significantly decreased (P = 0.0037, t-test) (E) whereas the density of DCX-positive cells is markedly increased (P = 0.014, t-test) (F). (n = 4 for Control, n =3 for cKO). Open arrowheads indicate respective cells.

G, H, I. Quantification two months after the induction of KO (n = 3 for Control, n = 4 for cKO). The density of RGL-ANSCs is not significantly different (P = 0.29, t-test) (G). The density of ANPCs is significantly decreased. (P = 0.0092, t-test) (H). The density of neuroblasts is not significantly different (P = 0.76, t-test) (I).

J, K, L. Quantification of proliferating cells three weeks after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.027, Mann Whitney test) (J), and that of Ki67+ANPCs is also significantly decreased. (P = 0.034, t-test) (K). The density of Ki67+ neuroblasts is significantly increased (P = 0.0011, t-test) (N) (n = 4 for Control, n = 4 for cKO) (L).

M, N, O. Quantification of proliferating cells two months after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.0086, t-test) (M), and densities of Ki67+ANPCs and Ki67+neuroblasts are also significantly decreased. (P = 0.023, P = 0.025, t-test) (n = 3 for Control, n = 3 for cKO) (N, O).

P. Declined return to RGL-ANSCs. Left, experimental scheme. Analysis was performed 3 weeks after BrdU injections. Middle, a representative image of BrdU+GFAP+ RGL-ANSCs. Right, Quantification of BrdU+GFAP+RGL-ANSCs (P = 0.019, n = 4 for Control, n = 4 for cKO).

Data information: Data presented as mean ± s.e.m. Scale bars, 100 µm in (B), 25 µm in (C, P) and 10 µm in (D, F, H). * P < 0.05, **P < 0.01, *** P < 0.001, t-test.
Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis (2020)
Figure 5

Figure 5. Lamin B1-knockout depletes adult neurogenesis in the long term.

A. Schematic of TAM treatment and collection of brain tissue.

B, C. Representative confocal images of the SGZ 6.5 months after the treatment with TAM.

D-I. Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (D) The density of RGL-ANSCs tends to be reduced in cKO. F (2, 8) = 3.98, P = 0.063, One-way ANOVA. (E) The density of ANPCs was reduced in cKO-8M and Cont-15M mice. F (2, 8) = 11.49, P = 0.0044, One-way ANOVA followed by Tukey's HSD test (** P < 0.01). (F) The density of DCX-positive cells was reduced in WT-old and cKO mice. p = 0.0091, Kruskal-Wallis test followed by Dunn's test (* P < 0.05). (G) The density of Ki67+RGL-ANSCs was reduced in cKO-8M mice (* P = 0.029, Kruskal-Wallis test followed by Dunn's test). (H) The density of Ki67+ANPC was not significantly different among three groups (P = 0.49, Kruskal-Wallis test). (I) The density of NeuN+EYFP+ neurons was reduced in cKO-8M mice. F (2, 8) = 34.85, P = 0.00011, One-way ANOVA followed by Tukey's HSD test (** P < 0.001). (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M).

Data information: Data represent mean ± s.e.m. Scale bars, 100 µm in (B), 50 µm in (C).
Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis (2020)
Figure 6

Figure 6. Lamin B1-dependent gene regulation in NPCs.

Schematic of Lmnb1-KO in NPCs. After the introduction of LV, NPCs were kept in proliferative conditions with FGF2 and EGF.

MA plot of differentially expressed genes between control and Lmnb1-KO NPCs.

C, D. Gene ontology analyses in LaminB1-regulated genes.

E. Fraction of direction of gene regulation by LaminB1. Genes were allocated dependent on where LaminB1 interacts with genes. Red bars indicated upregulated genes and blue bars indicate downregulated genes after lamin B1 knockout.

F. Examples of lamin B1-directed genes in NPCs. Blue bars indicate laminB1-LAD.
Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis (2020)
Figure 7

Figure 7. Exogenous expression of laminB1 represses neural differentiation

Experimental schema. After the introduction of RV into NPCs in a proliferative condition, FGF2 was withdrawn for 2 days, and samples were collected.

B, C. Confirmation of Lamin B1 overexpression by qRT-PCR (*P = 0.020, n = 3, one-sample t-test), and immunocytochemistry in NPCs. Scale bar = 10 µm.

D, Relative induced expression of differentiation markers upon the withdrawal of FGF2. 0 % indicates the same expression levels with control. Exogenous expression of lamin B1 inhibits the induction of genes related to neural differentiation (Tuj1, *P = 0.023; Prox1, ***P = 0.00012; NeuroD1, ** P = 0.0046; Ascl1, ***P = 0.00064; S100β, ***P = 0.000097; n = 3, one-sample t-test).

E, Experimental schema. RV:EGFP-IRES-EGFP or LaminB1-IRES-EGFP was injected into the DG of wildtype mice and the brains were collected 7 days later.

F, Confocal images after exogenous expression of lamin B1 in EGFP+ cells with RV;LaminB1-IRES-GFP (laminB1-OE.) Scale bar = 20 µm.

G, H, Exogenous expression of laminB1 repressed neuronal differentiation in vivo. An arrowhead indicate DCX+GFP+ cell in control (G, left) and open arrowheads indicate Sox2+GFP+ cells in lamin B1-OE cells. The fraction of EGFP+ DCX+ cells was significantly reduced by lamin B1 overexpression (***P < 0.0002, n = 3), whereas the fractions of EGFP+DCX+Sox2+ cells and the EGFP+Sox2+ were increased (EGFP+DCX+Sox2+, **P = 0.0024; EGFP+ Sox2+, P = 0.058; others, *P = 0.02). Scale bar = 20 µm.

Data information: Data represent mean ± s.d.

Expanded View Figure legends
Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis (2016)
Figure 1

Figure 1: CLU is required for proliferation and cycling of PC-3 cells.

(A) Cell proliferation for PC3 cells transfected with siSCR or siCLU monitored in real time for 60h with SP Dynamic Xcelligence system (Voltage 2.5 K). Error bars represent mean ± SEM, n=4. Slopes compared by ANCOVA test, p<0.0001. (B) Left panel: Cell cycle profile of PC3 cells transfected with siSCR or siCLU. Right panel: Percentage quantification of variation in G1, S and G2/M phases. Error bars represent mean ± SEM, n=3, **p<0.01 by paired Student's t-test. (G2/M population p= 0.0058; G1 p=0.008.). Inset: Western blot with CLU antibody in PC3 cells after CLU silencing. Vinculin was used as loading control. (C) Left panel: RNA-Microarray analysis comparing PC3 cells transfected with siCLU versus siSCR and Kinexus phosphokinome microarray analysis comparing LNCaP-transfected with siCLU versus siSCR. Target genes expression is shown as fold change relative to siSCR, p ≤0.05 by an unpaired t-test for all the genes listed. Central panel: Venn diagram to graphically illustrate the overlap of mRNA and proteins involved in the regulation of mitosis in both transcriptome and phosphokinome analysis. The comparison of these 2 experiments shows modification of 4 mitosis regulators. Right panel: Western blot validating the expression levels of target proteins.