EMBO/soda-nel huggingface dataset:
HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement (2021)
Figure 7

Figure 7 - hst-1 suppresses movement of endogenous miR395 and miR160, and alters xylem pole specification by mobile miR165/166

(A) miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.). U6 was probed as an endogenous control. Relative U6-normalized band-intensity quantifications are indicated for each tissue in -SO4 conditions.

(B) Stem-loop RT-qPCR-based analysis of rootstock/scion ratio of miR395a levels in plants grafted with hyl1-2 rootstocks onto scions of indicated genotypes (top) and RT-qPCR analysis of APS4miR395 mRNA levels in the corresponding hyl1-2 rootstocks (bottom), under -SO4 conditions. Error bars: SD. t-test (top) and Welch's t-test (bottom) p-values are indicated. n=4.

(C) miR160 northern analysis in Meselect-separated leaf vasculature and epidermis in WT versus hst-1 plants, in three biological replicates (rep.). U6: as in (A). Relative U6-normalized band-intensity quantifications are indicated.

(D) Stem-loop RT-qPCR-based analysis of epidermis/vasculature ratio of miR160 levels (top) and RT-qPCR analysis of ARF17miR160 mRNA levels (bottom) in the samples in (C). Error bars: SD. t-test (top) and Tukey's multiple comparisons test (bottom) p-values are indicated. n=3.

(E) Basic fuchsin staining of protoxylem (unfilled arrowheads) and metaxylem (filled arrowheads) in WT, shr-2 or hst-1 roots. *: protoxylem gap. Scale bars: 10 µm.

(F) miR165/166 northern analysis in WT versus hst-1 roots. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated.

(G) RT-qPCR analysis of pri-miR165a/-miR166a, SHR, SCR, PHBmiR165/166 and PHVmiR165/166 mRNA levels in WT versus hst-1 roots. Error bars: SD. t-test p-values are indicated. n=4.
The circadian oscillator analysed at the single-transcript level (2021)
Figure 1

Figure 1: Single-Molecule RNA Fluorescence In Situ Hybridisation (smRNA FISH) captures transcript distributions of core clock genes in mouse fibroblasts at multiple time points.

(A) smFISH targeting Bmal1, Cry1 and Nr1d1 in wild-type NIH 3T3 cells at 17h, 29h and 25h after synchronisation with Dex, respectively. Nuclei are stained with DAPI (blue). Each fluorescent dot (white) corresponds to a single transcript. Segmented cell boundaries are delineated in grey. The blue and white channels represent maximum z-projections. Scale bar: 20μm.

(B) Number of Bmal1, Cry1, and Nr1d1 transcripts per cell as a function of time after treatment with Dex. These data combine all smFISH hybridizations from the 4h sampled Nr1d1/Cry1 and Bmal1/Cry1 experiment. Each dot shows the average over one replicate from an independent slide (Methods). The solid lines represent fits using a two-harmonic cosinor model (Equation (1), Methods) for each gene individually.

(C) The inferred peak phase (angular component of the graph) and max-to-min fold change (radial component) from the fit of Equation (1).

(D) Distributions of Nr1d1, Cry1 and Bmal1 transcripts at 21h, 29h and 37h after synchronisation with Dex. μ represents the mean of the distribution, CV represent the coefficient of variation (standard deviation / mean), and N is the number of cells at the given time point. Total number of cells analysed for the Nr1d1/Cry1 pair: 21h - 449; 25h - 414; 29h - 521; 33h - 463; 37h - 477; 41h - 429. Total number of cells analysed for the Bmal1/Cry1 pair: 17h - 465; 21h - 490; 25h - 504; 29h - 436; 33h - 407; 37h - 454; 41h - 404.
The circadian oscillator analysed at the single-transcript level (2021)
Figure 2

Figure 2: Core circadian clock transcript distributions show area dependence and distinct gene-to-gene correlation structures.

(A) Log conditional mean mRNA count for a given area as a function of the log area. Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile. The conditional mean given the area E[m|A] is then the mean mRNA count of each bin. The area is normalised such that the average area is equal to one. Solid lines represent a least-squares linear fit to the data.

(B) Top image: Dual-channel smFISH targeting Bmal1 and Cry1 simultaneously, taken at 17h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Bmal1 transcripts; red, Cry1 transcripts. Bottom image: Dual-channel smFISH targeting Nr1d1 and Cry1 simultaneously, taken at 25h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Nr1d1 transcripts; red, Cry1 transcripts. Scale bar: 20μm.

(C) Bivariate distributions of mRNA counts per cell for dual-channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1. Each dot corresponds to a single cell, and the contours represent KDE estimates of the density. Time represents the number of hours after Dex synchronisation, and R represents the Pearson correlation coefficient.
CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane-induced alopecia (2019)
Figure 1

Figure 1 - Taxanes increase the number of phospho-histone H3+ cells in the anagen hair matrix

A-B) 100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of Ki-67+ cells (A) and EdU+ cells (B) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients. C) 100 nM paclitaxel treatment (24h) significantly (p= <0.0001 [****]) increased the number of mitotic phospho-histone H3 (pH3)+ cells in the hair matrix. Welch's t-test performed using N of 9 HFs from 3 patients. D) 100 nM docetaxel treatment (24h) significantly (p = 0.0004 [***]) increases the number of pH3+ cells in the hair matrix. Unpaired t-test performed using N of 8-9 HFs from 3 patients. E) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on (i) Ki-67 expression [paclitaxel]; (ii) EdU incorporation and pH3 immunoreactivity [paclitaxel]; (iii) pH3 immunoreactivity [docetaxel]. 20 µm scale. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.
CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane-induced alopecia (2019)
Figure 3

Figure 3 - Paclitaxel blocks nascent transcription and significantly increases cleaved caspase 3 immunoreactivity in hair matrix keratinocytes

A) Nascent RNA synthesis, as detected by ethynyl uridine (EU) incorporation, is blocked within clusters of nuclei in the hair matrix (arrows) following paclitaxel treatment (100 nM, 24h). 20 µm scale. B) Quantitative analysis highlights a significant (p =<0.0001 [****]) decrease in the number of EU+ nuclei following 24h paclitaxel treatment. Welch's t-test performed using N of 11-12 hair follicles (HFs) from 3 patients. C) Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3+ cell population that accumulates in response to paclitaxel treatment (see Figure 1). 10 µm scale. D) Cleaved caspase 3 expression in the hair matrix following 24h paclitaxel treatment. 20 µm scale. E) 100 nM paclitaxel treatment significantly (p = 0.0016 [exact] [**]) increases the number of cleaved caspase 3+ cells in the hair matrix after 24h. Mann Whitney U test performed using N of 16-18 HFs from 5 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.
CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane-induced alopecia (2019)
Figure 4

Figure 4 - Taxanes induce apoptosis and mitotic defects within human hair follicle K15+ epithelial stem/progenitor cell niches

Ai) Paclitaxel treatment promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase 3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF). 20 µm scale. Aii) High magnification montage demonstrating mitotic arrest (orange arrow) and apoptosis/caspase 3 positivity (white arrow) within the K15+ bulge. 10 µm scale.

B) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments (see methods). 50 µm scale. EC - extended cultures.

C) Graph showing significantly (p = 0.029 [**]) increased K15/caspase 3 double+ cells within the bulge following extended paclitaxel HF organ cultures. Welch's t-test performed using N of 8-9 HFs from 3 patients.

D) K15+ cells of the human HF bulge express Ki-67 during extended organ culture experiments. Paclitaxel treatment did not significantly affect the number of bulge K15/Ki-67 double+ cells. Unpaired t-test performed using N of 8-9 HFs from 3 patients.

E) Representative double immunofluorescence images of elevated γH2A.X immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments (see methods). 50 µm scale. EC - extended cultures.

F) γH2A.X analysis showing a significant (p = 0.065 [**]) increase in the number of cells with DNA double-strand breaks in the K15+ bulge following extended organ culture experiments. Mann Whitney U test performed using N of 5-6 HFs from 2 patients.

Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error. pbORS - proximal bulb outer root sheath.
CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane-induced alopecia (2019)
Figure 5

Figure 5 - Palbociclib blocks paclitaxel induced mitotic defects and apoptosis in the human hair follicle matrix

A) Schematic of experimental design. Hair follicles (HFs) were pre-incubated with the CDK4/6 inhibitor palbociclib for 18h, followed by a further incubation period with and without paclitaxel (or paclitaxel alone) for an additional 24h.

B) Palbociclib-only and palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki-67 expression in the hair matrix beyond that seen with just 24h treatment (see Appendix Figure S2B). Data also confirms that Ki-67 expression is unaffected by paclitaxel treatment (see also Figure 1A). Analysis performed using N of 6-9 HFs per condition from 3 patients. Ordinary one-way ANOVA with multiple comparisons performed. Adjusted p values = 0.0001[***] and 0.0001 [****] respectively.

C) Data confirming that paclitaxel treatment significantly increases (adjusted p value = 0.0001[***]) the number of pH3+ cells in the hair matrix (see also Figure 1C). This effect was not observed when paclitaxel treated hair follicles were pre- and co-incubated with palbociclib. Analysis performed using N of 6-9 HFs per condition from 3 patients

D) Cleaved caspase 3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment (see Figure 3D-E and results, main text) (adjusted p value = 0.0056 [**]). Paclitaxel treated hair follicles pre- and co-incubated with palbociclib do not show enhanced cleaved caspase 3 expression, contrasting with paclitaxel-only treatment. Ordinary one-way ANOVA with multiple comparisons performed using N of 4-5 HFs per condition.

E) Immunofluorescence data representing how palbociclib and/or paclitaxel affect (i) Ki-67 expression, (ii) pH3 immunoreactivity and (iii) cleaved caspase 3 expression. 20 µm scale.

Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.
CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane-induced alopecia (2019)
Figure 6

Figure 6 - Transient CDK4/6 inhibition emphasises the cell cycle dependency of paclitaxel cytotoxicity in the human hair follicle

A) Schematic of experimental design. Hair follicles (HFs) were pre-treated with palbociclib for 18h then further incubated either with or without paclitaxel for a further 24h. Subsequently, drug-containing media was washed out and HFs were left in organ culture for an additional 24-48h period.

B) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle. DNA synthesis in the hair matrix resumed in the palbociclib-only and palbociclib plus paclitaxel dual treated conditions following washout (see also Appendix Figure S3). Ordinary one-way ANOVA with multiple comparisons performed using N of 8-13 HFs from 3 patients.

C) A trending increase in the number of cleaved caspase 3+ cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel-only treatment significantly increased the number of cleaved caspase 3+ cells in the hair matrix (adjusted p value = 0.0003 [***]). Palbociclib-only treatment did not increase the number of cleaved caspase 3+ cells in the hair matrix following drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Caspase 3 vehicle vs. paclitaxel data are also used in Appendix Figure S1E.

D) HFs treated with paclitaxel alone showed a significant (adjusted p value = 0.0316 [*]) increase in the number of pH3+ cells in the matrix after washout. A significant (adjusted p value = 0.0278 [*]) increase in the number pH3+ cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3+ hair matrix keratinocytes after drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. pH3 vehicle vs. paclitaxel data are also used in Appendix Figure S1B.

E) Representative fluorescence images of EdU and pH3/cleaved caspase 3 staining in paclitaxel-only and palbociclib plus paclitaxel dual treated HFs following drug washout. Where DNA synthesis has resumed following reversal of the G1 arrest, paclitaxel cytotoxicity is emergent.

Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error. Please also see supporting data in Appendix Figure S3 and S4. HS - hair shaft; "wo" - washout. 20 µm scale.
CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane-induced alopecia (2019)
Figure 7

Figure 7 - CDK4/6 inhibition antagonises paclitaxel cytotoxicity in the stem/progenitor cell-rich human hair follicle outer root sheath

A) Schematic of experimental design. Hair follicles (HFs) were pre-treated with (or without) palbociclib for 18h then further incubated either with/without paclitaxel for a further 24h. Subsequently, drug-containing media was washed out and HFs were cultured for an additional 24-48h period.

B) Paclitaxel treatment did not affect the number of EdU+ cells within the HF outer root sheath (ORS) following washout. In contrast, palbociclib treated HFs lacked EdU incorporation following drug washout (adjusted p value = 0.0001 [***]). The number of EdU+ cells was significantly decreased in the outer root sheath (ORS) of HFs dual treated with both paclitaxel and palbociclib (adjusted p value = 0.0021 [**]). Ordinary one-way ANOVA with multiple comparisons performed using N of 12-13 HFs from 3 patients.

C) HFs treated with paclitaxel showed a significant (adjusted p value = 0.0001 [****]) increase in the number of cleaved caspase 3+ cells in the ORS following drug washout. Palbociclib-only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3+ ORS cells. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Data for vehicle vs. paclitaxel are utilised also in Appendix Figure S1H.

D) Paclitaxel-only treated HFs showed a significant (adjusted p value = 0.0001 [****]) increase in the number of pH3+ ORS cells following drug washout. Palbociclib treatment alone had no significant effect on the number of pH3+ ORS cells. There was a trending, yet non-significant, increase in the number of pH3+ ORS cells in HFs dual treated with palbociclib plus paclitaxel. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Data for vehicle vs. paclitaxel are utilised also in Appendix Figure S1I.

E) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout, whereby DNA synthesis has not resumed. F) Representative images of pH3/caspase 3 staining within the ORS of paclitaxel-only and palbociclib plus paclitaxel treated HFs following drug washout. Please see supporting data in Appendix Figure S7.

G-H) Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of cleaved caspase 3+ (G) and pH3+ (H) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. K15/pH3 pbORS paclitaxel-only treatment HF image (H, bottom left panel) can be seen at a higher magnification in Figure 4Ai.

Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. E-F 50 µm scale; G-H 20 µm scale.
The Spectrin cytoskeleton regulates the Hippo signalling pathway. (2015)
Figure 1

The Spectrin cytoskeleton restricts tissue growth in the Drosophila eye and wing

A-O UAS.RNAi lines were driven with eyeless.Gal4 gmr.Gal4 for expression during eye development or nubbin-Gal4 for expression during wing development. (A, B) Control adult Drosophila eye (A) and wing (B). (C, D) α-spectrin RNAi results in overgrowth of the eye (C) and wing (D). (E, F) βH-spectrin/karst RNAi results in overgrowth of the eye (E) and wing (F). (G, H) β-spectrin RNAi does not affect eye size (G) or wing size (H). (I, J) kibra RNAi results in overgrowth of the eye (I) and wing (J). (K, L) α-spectrin, kibra double RNAi results in stronger overgrowth of the eye (K) and wing (L). (M, N) βH-spectrin/karst, kibra double RNAi results in stronger overgrowth of the eye (M) and wing (N). (O) Quantification of female wing sizes by pixel area, 5 wings per genotype were measured. Error bars show standard deviation.

P-RThe eyeless FLP MARCM system was used to generate clonally mutant fly eyes. kibra mutant eyes (Q) overgrow slightly compared to controls (P), while kibra mutant eyes expressing α-spectrin RNAi (R) overgrow strongly compared to controls (P).

Data information: Scale bars, 250 μm.
The Spectrin cytoskeleton regulates the Hippo signalling pathway. (2015)
Figure 2

The Spectrin cytoskeleton represses interommatidial cell number and Hippo target gene expression in parallel with Kibra

A-F The eyeless FLP MARCM system was used to generate mutant eyes that also express UAS.RNAi targeting the Spectrin cytoskeleton. Pupal retinas were examined at 42–46 h after puparium formation (APF). Cell membranes were marked with Dlg staining. (A) Control pupal retina showing cone cells surrounded by interommatidial cells. (B) kibra mutant pupal retina displaying additional interommatidial cells. (C) Pupal retina expressing α-spectrin RNAi showing additional interommatidial cells. (D) kibra mutant pupal retinas expressing α-spectrin RNAi showing many additional interommatidial cells. (E, F) Clones of α-spectrin mutant cells (GFP negative) (E) and βH-spectrin/karst mutant cells (GFP negative) (F) show extra interommatidial cells. Scale bars, 20 μm.

G-K UAS.RNAi lines were driven with hh.Gal4 UAS.GFP for expression in the posterior compartment and contained the ex.lacZ reporter transgene. (G) Control wing showing a normal ex.lacZ expression pattern, which is low at the dorsal–ventral boundary but high in the proximal regions of the wing disc. (H) RNAi knock-down of α-Spectrin results in a mild up-regulation of ex.lacZ in the posterior compartment. (I) RNAi knock-down of kibra results in a mild up-regulation of ex.lacZ in the posterior compartment. (J) Dual RNAi knock-down of both α-spectrin and kibra results in enhanced up-regulation of ex.lacZ in the posterior compartment. (K) RNAi knock-down of hippo results in an up-regulation of ex.lacZ in the posterior compartment. Scale bars, 50 μm.
The Spectrin cytoskeleton regulates the Hippo signalling pathway. (2015)
Figure 3

The apical α-βH Spectrin cytoskeleton binds to and co-localises with Expanded protein and acts genetically upstream or at the level of Expanded to control wing growth

A Co-IP of V5-tagged Expanded with the FLAG-tagged N-terminal region of Karst/βH-Spectrin, but not other Karst truncation constructs.

B, C Expanded co-localises with Karst at the apical membrane of wing imaginal disc cells (apical view, B; cross section, C).

D Control nubbin.Gal4 expressing wing.

E Overexpression of Ex activates Hippo signalling to produce a small wing.

F RNAi knock-down of α-Spectrin results in an overgrown wing.

G Overexpression of Ex blocks the effect of α-Spectrin RNAi.

H RNAi knock-down of βH-spectrin/karst results in an overgrown wing.

I Overexpression of Ex blocks the effect of βH-spectrin/karst RNAi.

Data information: Scale bars, 10 μm (B, C), 250 μm (D–I).
The Spectrin cytoskeleton regulates the Hippo signalling pathway. (2015)
Figure 4

The apical α-βH Spectrin cytoskeleton may be mechanosensory in the wing imaginal disc

A Schematic diagram of central compression and circumferencial stretching in the third instar wing pouch.

B-D Third instar wing imaginal disc stained for beta-galactosidase expressed from the expanded.lacZ reporter gene (B), Crb (C) and Kst-YFP (D). Quantification of line intensity shown below. Note inverse correlation of Crb and Kst-YFP with ex.lacZ. Scale bars, 50 μm.

E, F Zoom of Kst-YFP cells under compression (E) and under stretch (F). Scale bars, 10 μm.

G Schematic diagram of the effect of force on the apical spectrin cytoskeleton leading to declustering of Crumbs complexes and reduced Hippo signalling.
The Spectrin cytoskeleton regulates the Hippo signalling pathway. (2015)
Figure 5

CrbEx™-GFP restricts tissue growth and acts upstream of Wts and in parallel with Ajuba

A Schematic diagram of clustering of Crb complexes by expression of CrbExTM-GFP.

B Control nub.Gal4 wing.

C, D Expression of UAS.CrbExTM-GFP with nub.Gal4 (C) or UAS.Wts with nub.Gal4 (D) results in a small wing.

E RNAi knock-down of α-Spectrin results in an overgrown wing.

F, G Expression of UAS.CrbExTM-GFP (F) or UAS.Wts (G) blocks the effect of α-Spectrin RNAi.

H RNAi knock-down of βH-spectrin/karst results in an overgrown wing.

I, J Expression of UAS.CrbExTM-GFP (I) or UAS.Wts (J) blocks the effect of α-Spectrin RNAi.

K, L RNAi knock-down of Wts results in a strongly overgrown wing (K) and expression of UAS.CrbExTM-GFP does not block the effect of wts RNAi (L).

M, N RNAi knock-down of Ajuba results in a small wing (M), and expression of UAS.CrbExTM-GFP enhances the effect of ajuba RNAi (N).

O Quantification of various wing sizes. Error bars show standard deviation.

P, Q Localisation of Wts-GFP (P) and E-cadherin (Q) in the third instar wing imaginal disc.

R Quantification of line intensity in (P) and (Q). No increased Wts-GFP intensity is observed in proximal regions where cells are subject to stretching.

Data information: Scale bars, 250 μm (B–N), 40 μm (P, Q).