EMBO/soda-nel huggingface dataset:
Novel analogue of colchicine induces selective pro-death autophagy and necrosis in human cancer cells. (2014)
Figure 4

Green 1 Induces Selective Pro-Death Autophagy.

(A) Pancreatic cancer cells, human leukemia cells (Jurkat) and NHFs were incubated with Monodansylcadaverine (MDC) to stain autophagic vacuoles and propidium iodide (PI) for cell death to determine if autophagic induction led to cell death. Bright, punctate staining is indicative of autophagy, as seen in our positive control (Tamoxifen treated cells). Images were taken at 400X magnification on a fluorescent microscope. (B) Western blot analyses of Beclin-1 expression and LC3 conversion were conducted on PANC-1 cells treated with 5.0 µM Green 1 at various time points. β-actin was measured as an internal control. Samples treated for 3 and 6 hours were compared to the early control, while the sample treated for 48 hours was compared to the 48-hour control. Results are expressed as mean ± SD from two independent experiments for both Western blots.
Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina and human retina (2016)
Figure 1

Figure 1. The red-shifted channelrhodopsin ReaChR can be efficiently targeted to the RGC membrane and dendritic arbor of blind mice.

(A) In vivo fundus image of an AAV-injected rd1 mouse expressing ReaChR-mCitrine driven by the hSyn promoter (AAV2-hSyn:ReaChR-mCitrine); transduced RGCs are visible as green puncta. (B) Live 2-photon image showing that ReaChR-mCitrine expression is restricted to RGC-membranes and their dendritic processes. (C) Confocal image of a vertical retinal section expressing ReaChR-mCitrine. Endogenous mCitrine (mCit) fluorescence, is localized to RGC membranes as well as RGC dendritic processes (see also Fig EV1A); blue: DAPI (to visualize retinal nuclei); inner nuclear layer (INL); inner plexiform layer (IPL) and ganglion cell layer (GCL); scale bars = 10 µm.
Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina and human retina (2016)
Figure 2

Figure 2. Red-shifted optogenetic stimulation of ReaChR triggers responses in the retinae of blind mice injected with AAV2-hSyn:ReaChR-mCitrine.

(A) Light sensitivity: Photocurrents of a ReaChR-expressing RGC stimulated with varying light intensities at 590 nm (~1013 − 1018 photons cm-2 s-1). (B) ReaChR-induced photocurrents as a function of light intensity (590 nm, n = 7 cells, mean photocurrent amplitude at 4.2 × 1017 photons cm-2 s-1 was −349 ± 192 pA). At the bottom, the sensitivity range for wild-type cones (Nikonov et al, 2006) is shown in comparison to the sensitivity range of ReaChR expressing RGCs. The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm, respectively (EU-Directive, 2006; ICNIRP, 2013). (C) Photocurrent action spectrum of a ReaChR-expressing RGC (1.2 × 1016 photons cm-2 s-1). (D) Normalized action spectrums of ReaChR-RGCs. Solid line represents patch-clamp data (n = 8 cells; 1.2 × 1016 photons cm-2 s-1). The mean photocurrent amplitude at 550 nm was −324 ± 97 pA. Dashed line represents multi-electrode array data (n = 168 cells; ~1016 photons cm-2 s-1). (E) Temporal properties. Modulation of ReaChR-induced photocurrents at increasing stimulation frequencies (patch-clamp recording from a ReaChR-expressing RGC; at 22 Hz magnified trace is shown; 1.3×1016 photons cm-2 s-1). (F) Spiking modulation ratio of ReaChR-RGCs quantified from multi-electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz (297 cells, 1.3 × 1016 photons cm-2 s-1). Graphs from (B), (D) and (F) are presented as mean ± SEM.
Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina and human retina (2016)
Figure 3

Figure 3. ReaChR-triggered responses in blind rd1 mice, treated with AAV2-hSyn:ReaChR-mCitrine can be detected in the visual cortex at a range of orange light intensities below the safety threshold.

(A) Examples of local field potential (LFP) traces in response to light flashes at 595 nm (light stimulus is shown as orange bar; 1.25 × 1017 photons cm-2 s-1). (B) Spike raster plots in response to 200 repetitions of the same light stimulus. (C) Averaged spike responses shown as peristimulus time histograms (PSTH, bin size: 5 ms) based on the spike raster plots. Top-row: WT-mice, middle row: rd1 control mice, bottom-row: ReaChR-treated rd1 mice. (D) ReaChR-induced cortical responses (spikes and LFP) as a function of light intensity at (595 nm) demonstrating that ReaChR-treated rd1 mice (n = 4) can detect light in the visual cortex at intensities well below the safety threshold allowed for the human retina. Data presented as mean ± SEM.
Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina and human retina (2016)
Figure 4

Figure 4. ReaChR-evoked responses are sufficient to modulate the behavior of blind mice treated with AAV2-hSyn:ReaChR-mCitrine.

(A) Locomotory behavior of a representative rd1 and ReaChR-treated mouse during illumination with light at 590 nm in an open field arena (irradiance measured at the level of the mice's eyes was ~1015 photons cm-2 s-1, cumulative activity trajectories are shown as red traces after 2 min of illumination), see also EV Movie 1 and EV Movie 2. (B) Mean velocities of the ReaChR-treated and rd1 mice analyzed over a duration of 2 min at light onset. The mean velocity of ReaChR-treated mice were significantly reduced than rd1 mice (P = 0.006, one-tailed t-test) upon illumination. Mean ± SEM are shown for each group. ReaChR-treated rd1 (n = 11), rd1 (n = 12). (C) Light/dark box test. Percentage of time mice spent in the light compartment as a function of light intensity (WT, n = 9; rd1, n = 9; ReaChR-treated n = 7). Mean ± SEM are shown for each group. (D) Percentage of time rd1 (n = 9), WT (n = 9) and ReaChR-treated (n = 7) mice spent in the light compartment for an illumination intensity of 2.11 × 1015 photons cm-2 s-1. Both WT as well as ReaChR-treated mice spent significantly less time in the light compartment than the rd1 mice (P < 0.0001, ordinary one-way ANOVA). *** and **** indicates significance levels determined by Tukey's multiple comparisons test for rd1 vs. WT and rd1 vs. ReaChR-treated. See also EV Movie 3 for the response of a representative ReaChR-treated rd1 mouse plotted in (D).
Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina and human retina (2016)
Figure 5

Figure 5. Evaluation of the translational potential of ReaChR-expression and light responses in AAV-infected macaque retinae.

(A) Vertical section of macaque retinal explant transfected with AAV2 hSyn:ReaChR-mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL. (B) Vertical section of macaque retinal explant transfected with AAV8 Y447 733F-CAG:ReaChR-GFP after 3 days of incubation. GFP expression was primarily observed in the cell bodies but also in processes of RGCs; green: endogenous GFP fluorescence; blue: DAPI; outer nuclear layer (ONL). scale bars = 10 µm. (C) Averaged spike responses obtained from multi-electrode array recordings shown as PSTH of 4, 8 and 2 cells respectively of three different macaques (580 nm; 1.24 × 1017 photons cm-2 s-1). PSTHs are presented as mean ± SEM (dashed lines, calculated over responding cells). Raster plots of a representative cell from each individual macaque are shown below (10 stimulus-repetitions). (D) Action spectrum of ReaChR in primate RGCs from multi-electrode array recordings. Data is shown as mean ± SEM of the normalized firing rate, averaged from three individual macaques (28 cells, error bars calculated over cells), see also Fig EV2B for action spectrums of three individual macaques obtained from multi-electrode array recordings. (E) Responses (Hz) of a ReaChR-expressing RGC (patch clamp recording, in cell-attached mode) to flicker stimulations at increasing frequencies (2−22 Hz; 1.3 × 1016 photons cm-2 s-1).
Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina and human retina (2016)
Figure 6

Figure 6. In the human retina, ReaChR is functional in ganglion cells of the para-fovea at light intensities safe for the human eye in a macular explant transduced with lentivirus encoding hSyn:ReaChR-mCitrine.

(A) Schematic illustrates the size and location of the retinal explant (dashed line). (B) Brightfield image of the retinal explant showing the macula (yellow pigmented) with the approximate location of the multi-electrode array chip indicated. (C) Infrared image of the explant pressed against the multi-electrode array; the black dashed line depicts the 'electrophysiological boundary' of the para-fovea based on the absence of RGC-spike trains (scale bar: 100 µm; see also Fig EV3A and B). Voltage trace of a para-foveal RGC stimulated with orange light (600 nm; 1.1 × 1017 photons cm-2 s-1) is shown on the right. Individual electrodes that recorded ReaChR-responding cells are indicated in green. (D) Action spectrum of ReaChR constructed from (6 cells, mean ± SEM, error bars calculated over cells) exhibiting peak activity at 550 nm (1.1 × 1017 photons cm-2 s-1) similar to what was observed in mice and in the macaque retinae. (E) Peristimulus time histograms constructed for wavelengths 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate ReaChR-evoked spike trains (under L-AP4) in the human retina (6 cells, mean ± SEM, error bars calculated over cells), bin size: 100 ms; Bottom: Raster plots of the light responses of a representative spike-sorted cell.
Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina and human retina (2016)
Figure 7

Figure 7. In the human retina, ReaChR was expressed in para-foveal RGCs as well as in near-peripheral RGCs when a macular explant was transduced with a lentivirus encoding hSyn:ReaChR-mCitrine.

(A) Confocal image of the central retina, exhibiting endogenous ReaChR-mCitrine fluorescence (mCit) in ganglion cells, located within ~500 µm from the center of the foveal pit, a part of the human retina where midget cell density is estimated to be ~95% of total RGC population (Dacey, 1993) (arrowhead: soma; arrow: dendritic arbor). The dashed line illustrates the beginning of the foveal slope leading towards the ‘cone-only’ foveal pit. (B) Confocal image of the same explant at a higher retinal eccentricity (3-4 mm from the foveal center) showing ReaChR-mCitrine fluorescence in RGCs with larger dendritic arbors. The endogenous mCitrine was not amplified. Scale bars: 10 µm. PR: Photoreceptors, GC: Ganglion cells, DAPI (blue).
Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina and human retina (2016)
Figure 8

Figure 8. AAV mediated optogenetic activation of the human retina.

(A) Human retinal explant, prepared from the far periphery, infected with an AAV8 Y447 733F-CAG :ReaChR-GFP, placed on a multi-electrode array chip (left); GFP epifluorescence of the same explant following 12 days of incubation is shown on the right. Scale bars: 100 µm. The location of two cells that responded to repeated 2 s full field stimulations of orange light (600 nm) are highlighted. (B) The corresponding voltage traces show that the responses in the far peripheral human retina are more transient, compared to the experiments from the human para-fovea (see Fig 6C and E, Fig EV3B) where only sustained light responses were evoked. The light response of cell (2) was restricted to a burst of action potentials fired within the initial ~300 ms in response to a 2 s full field 600 nm light stimulus. (C) Peristimulus time histograms constructed for wavelengths from 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate AAV-ReaChR-evoked spike trains (mean ± SEM, error bars calculated over 3-4 cells, bin size: 100 ms) Bottom: Raster plots of the light responses of a representative spike-sorted cell. All recordings were performed under pharmacological blockade of photoreceptor input (50 µM L-AP4).
Loss of ANCO1 repression at AIB1/YAP targets drives breast cancer progression (2019)
Figure 1

Figure 1. AIB1 and YAP coregulate a subset of TEAD target genes.

A) AIB1 and YAP co-activate and corepress a subset of TEAD target genes. Venn diagrams showing differentially regulated genes in MCF10A cell lines that overexpress YAP or YAP S94A in the presence of control shGFP or shAIB1 as indicated. RNA-sequencing performed in triplicate.

B) Heatmaps of AIB1-YAP co-activated, YAP activated, AIB1-YAP corepressed, and YAP repressed genes shown in panel (A). Red and blue indicate high and low levels, respectively.

C) Select genes are regulated by YAP and AIB1, others are YAP regulated independently of AIB1. Endogenous gene expression changes assessed by RT-qPCR.

D) Significant genomic co-occupancy between AIB1 and TEAD in normal breast and breast cancer cells. Overlap of AIB1 and TEAD4 ChIP-seq peaks in MCF10A and MCF7 cell lines. Select peaks from MCF10A ChIP-seq shown. ChIP-sequencing performed in triplicate.

E) AIB1 and TEAD are localized at promoters and enhancers globally. Feature distribution based on overlapping AIB1-TEAD4 peaks with respective MCF10A and MCF7 histone ChIP-seq peaks.

F) TEAD binding motifs are enriched within AIB1 occupied sites across the genome. HOMER de novo motif analysis of AIB1 ChIP-seq peaks in MCF10A and MCF7 cells.

G) AIB1 modulates TEAD transcriptional activity in a YAP dependent manner. HEK293T cells were transfected with CTGF-promoter reporter and expression vectors of indicated proteins.

H) AIB1 is engaged with TEAD on co-activated target promoters in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.

I) AIB1 is engaged with TEAD on co-activated target promoters in early stage breast cancer cells. Sequential ChIP-reChIP performed with indicated antibodies in MCFDCIS cells. Levels of precipitated chromatin assessed by qPCR.

Data information: RNA-sequencing was performed in triplicate and statistics in Panel (A-B) were calculated by EdgeR with Benjamini-Hochberg multiple test correction in R. ChIP-sequencing was performed on triplicates and statistics were calculated by random permutation of the genomic locations of ChIP peaks using the shuffle command in ChIPseeker in R. Student's t-test was used for the statistical analysis in Panels (C, H, and I); data represents the means+SD from at least 3 biological replicates. ANOVA with Tukey's post-hoc was used for the statistical analysis in Panel (G); data represents the mean+SD from at least 3 replicates (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=Not Significant, #=significant regulation in opposite direction).
Loss of ANCO1 repression at AIB1/YAP targets drives breast cancer progression (2019)
Figure 2

Figure 2. AIB1 recruits ANCO1 to YAP-TEAD to repress transcription at 1q21.3.

A) ANCO1 is recruited to YAP-TEAD by AIB1. Positive Proximity Ligation Assay (PLA) signal (white foci, falsely colored 594nm signal) shown between ANCO1-TEAD, ANCO1-YAP, AIB1-TEAD, and AIB1-YAP, in control cells are lost in AIB1 knockdown. Representative images of Proximity Ligation Assay (PLA) between indicated proteins in MCF10A shGFP (control) and shAIB1 cell lines using antibodies (species) as indicated; Scale bars shown indicate 25μm.

B) YAP overexpression represses, AIB1 knockdown dysregulates, and ANCO1 knockdown amplifies genes at the 1q21.3 locus in MCF10A and MCFDCIS cells. Gene set enrichment analysis of RNA-seq data and cDNA array data generated from MCF10A cell lines as indicated.

C) YAP represses 1q21.3 localized genes. Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP/S94A; levels were assessed by RT-qPCR.

D) ANCO1 knockdown increases expression of S100 and SPPR genes in MCF10A and MCFDCIS cells; levels were assessed by RT-qPCR.

E) YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown. Levels of endogenous 1q21.3 genes were assessed in YAP or S94A-expressing, control or shANCO1 MCF10A cells by RT-qPCR.

F) TEAD is engaged at annotated enhancer regions within 1q21.3. Analysis of TEAD4 ChIP-seq in MDA-MB-231(GSE66081), MCF7, and A549 Cells (GSE32465) at 1q21.3 with enhancers scored by GeneHancer. Enhancer regions of interest indicated between dotted lines.

G) TEAD, YAP, AIB1, and ANCO1 are engaged at annotated enhancer regions within 1q21.3. Chromatin Immunoprecipitation (ChIP) performed with indicated antibodies in MCF10A cells.

H) AIB1 is engaged with TEAD on superenhancer sites at 1q21.3 in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.

I) ANCO1 is recruited to superenhancers at 1q21.3 by AIB1 and is significantly reduced at 1q21.3 following AIB1 knockdown. ChIP performed with ANCO1 antibodies in control or shAIB1 cells. Levels of precipitated chromatin assessed by qPCR.

Data information: GSEA Enrichment Score and FDR used in Panel (B). Student's t-test was used for the statistical analysis in Panels (C, D, E, G, H, and I); data represents the means+SD from at least three biological replicates. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=Not Significant).
Loss of ANCO1 repression at AIB1/YAP targets drives breast cancer progression (2019)
Figure 3

Figure 3. AIB1 and ANCO1 regulate YAP induced invasion in vivo.

A) YAP driven aberrant sphere formation of MCF10A cell lines is ablated by AIB1 knockdown, but further enhanced by ANCO1 knockdown. Indicated cell lines were plated on Matrigel and allowed to form mammospheres for 7 days. 10x and 40x magnifications shown, Scale bar = 200μm.

B) Quantification of aberrant spheres per field displayed in panel (A), n= number of spheres.

C) Quantification of size (arbitrary units) of aberrant spheres per field displayed in panel (A), relative to control YAP S94A shGFP spheres. n= number of spheres.

D) MCFDCIS xenograft tumors have increased expression of AIB1-YAP, EMT, and 1q21.3 genes during the invasive transition. Schematic of experiment shown. Gene set enrichment analysis of RNA-seq data generated from MCFDCIS xenografts at indicated time points.

E) Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions. Representative images of H&E, p63, and ANCO1 stained MCFDCIS xenografts as DCIS and invasive lesions. Yellow circles indicate DCIS lesions and black arrows indicate invasive lesions. 20x magnification; Scale bar = 100μm.

F) Quantification of ANCO1 positivity in DCIS and Invasive lesions shown in panel (E).

Data information: Number of spheres quantified in Panels (B-C) described above, with middle lines and error bars indicating mean+SEM. ANOVA with Tukey's post-hoc was used for the statistical analysis in Panel (B). Kruskal-Wallis with Dunn's Multiple Comparison (non-paramatric test used because data was non-normally distributed following ImageJ quantification and thresholding) in Panel (C). GSEA Enrichment Score and FDR used in Panel (D). Student's t-test was used for the statistical analysis in Panel (F), with n > 3 mice per group, and with box limits denoting the range of data, and the middle line at the median (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=Not Significant).
Loss of ANCO1 repression at AIB1/YAP targets drives breast cancer progression (2019)
Figure 4

Figure 4. Loss of ANCO1 in breast cancer perturbs ANCO1 dependent YAP repression and is a marker for poor prognosis.

A) ANCO1 levels decrease in breast cancer. ANCO1 mRNA expression from normal tissue (GTEX) and Tumor (TCGA) for breast samples.

B) Representative IHC images of ANCO1 staining in normal human breast and DCIS lesions. DCIS lesions can be stratified to either ANCO1 negative (center panel), ANCO1 positive (right panel). Scale bar = 50μm.

C) High ANCO1 levels are a good prognostic indicator in Basal Breast Cancer in patients. KM plot showing progression free survival in patients with stratified ANCO1 expression. n= 360 patients.

D) Patient breast tumor samples with reduced ANCO1 levels have high expression of 1q21.3 genes, without altered AIB1-YAP co-activated gene expression. Dot plots showing TCGA Pan-Cancer data accessed via Xenabroswer. Patients were stratified to high or low expression of ANCO1 by median expression.

Data information: Student's t-test was used for the statistical analysis in Panel (A) (n = 179 in normal group and n= 1213 in tumor group). KMplot Statistics were calculated by KMplotter in Panel (C) (n = 360 patients). In panel (D), r values were calculated for each correlation (for individual correlations, p<0.001), and p values were determined for linear regression between groups (n > 150 patients). ****p<0.0001.
SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation (2021)
Figure 1

Figure 1 - KCC4 expression correlates with SIRT7 expression, and KCC4 activity is modulated by acetylation.

A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population (www.genenetwork.org). Adr: Adrenal Gland mRNA, Bla: Bladder mRNA, Bone: Bone Tibia mRNA, CNS: CNS mRNA, Cec: Cecum mRNA, Epi: Epididymis mRNA, Eso: Esophagus mRNA, Eye: Eye mRNA, Fat: Peritoneal Fat mRNA, GI: GI Track mRNA, Hea: Heart mRNA, Kid: Kidney mRNA, Liv: Liver mRNA, Lung: Lung mRNA, Mus: Gastrocnemius Muscle mRNA, Ova: Ovary mRNA, Pro: Prostate mRNA, Sal: Salivary Gland mRNA, Skin: Skin Back mRNA, Sn: Sciatic Nerve mRNA, Spl: Spleen mRNA, Tes: Testis mRNA, Thy: Thymus mRNA, Ton: Tongue mRNA, Tra: Trachea mRNA, and Ute: Uterus mRNA.

B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population (www.genenetwork.org).

C Oocytes injected with cRNA encoding KCC4 were incubated with NAD+ 200 μM and NAM 10 mM for 4 h. Lysates were immunoprecipitated with an anti-KCC4 antibody and then analyzed by SDS-PAGE/immunoblotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-injected oocytes (UI) were used as a control.

D Oocytes expressing KCC4 were incubated with NAM for the indicated times, and KCC4 protein levels (upper panel), and activity (lower panel) were analyzed by SDS-PAGE/immunoblotting or Cl--dependent 86Rb+ uptake under hypotonic conditions.

E Oocytes injected with H20 or KCC4 were incubated for the indicated times, and KCC4 protein levels were analyzed by SDS-PAGE/immunoblotting.

F Oocytes were injected with either WT, K114R or K114Q KCC4 and incubated with vehicle (V), NAD+ or NAM for 48 h, and KCC4 protein levels (upper pannel), and activity (lower pannel) were analyzed as in D.

Data information: Data are presented as mean ± SEM. RU: relative units. Depicted blots are representative of three independent biological replicates and values shown are the mean of the three experiments. To evaluate KCC4 activity, we performed three independent biological experiments with oocytes obtained from different frogs and each experiment included at least 4 replicates. * Indicates a significant difference vs time 0, at p<0.05 by Student's t-test.