EMBO/soda-nel huggingface dataset:
Lysosome membrane lipid microdomains: novel regulators of chaperone‐mediated autophagy (2006)
Figure 8

Lysosomes containing a mutant LAMP‐2A unable to move into discrete lysosomal membrane regions show enhanced CMA activity. (A) Lysosomes from mouse fibroblasts transfected with wild‐type hLAMP‐2A and maintained in the presence (Serum+) or absence (Serum−) of serum were collected by centrifugation and subjected to Triton X‐114 extraction, sucrose density gradient centrifugation and immunobloting for human (hL2A) or mouse (mL2A) LAMP‐2A. Lane 1: homogenate. (B) Amino‐acid sequence of the transmembrane and cytosolic region of hLAMP‐2A and the mutations performed. Replaced amino acids are shadowed. (C) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP‐2A and maintained in the presence of serum were processed as in (A). (D) The percentage of total lysosomal hLAMP‐2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here. Values, corrected for the amount of mLAMP‐2A in each fraction, are mean+s.e. of 3–4 experiments. (E) Changes in hLAMP‐2A distribution induced by serum removal. Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments. Changes in the three nonDR regions are shown together (no DR). (F) Proteolysis of a pool of 3[H]labeled cytosolic proteins by intact (left) or disrupted (right) lysosomes isolated from untransfected mouse fibroblasts (none) or fibroblasts transfected with wild‐type (WT) or the indicated mutant forms of hLAMP‐2A. Values are the mean+s.e. of two experiments with triplicate samples. (G) Part of the lysosomes from (F) was treated with MBCD before the incubation with the radiolabeled proteins. Values are expressed as percentage of degradation by untreated lysosomes and are the mean+s.e. of two experiments with triplicate samples.
Lysosome membrane lipid microdomains: novel regulators of chaperone‐mediated autophagy (2006)
Figure 9

Organization of LAMP‐2A in different regions of the lysosomal membrane. (A) The Triton X‐114‐resistant and soluble regions of the lysosomal membrane were subjected to centrifugation through a continuous (10–80%) sucrose density gradient, SDS–PAGE and immunoblot for LAMP‐2A. The molecular weight range of each fraction is as follows: fraction 1 (100 kDa); fraction 2 (100–200 kDa); fraction 3 (200–400 kDa); fraction 4 (400–600 kDa); fraction 5 (>600 kDa). (B) Fed rat liver lysosomes were incubated with 1 mM CaCl2 (+Ca2+) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors (PI). Samples were subjected to Triton X‐114 extraction, sucrose density gradients centrifugation and LAMP‐2A immunoblot. (C) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS–PAGE and immunobloted with antibodies against the cytosolic (Top; L‐2A) and luminal (Bottom; L‐2) regions of LAMP‐2A. Arrow: LAMP‐2A proteolytic fragment (Cuervo and Dice, 2000a). (D, E) Rat liver lysosomes incubated or not with 1 mM CaCl2 (+Ca2+) were subjected to Triton X‐114 extraction and sucrose density gradients centrifugation. Levels of cathepsin A (D) and carboxypeptidase A activity (E) were measured in each fraction by immunoblot or using a standardized colorimetric assay, respectively. Values in (E) are expressed as times the activity in untreated membranes and are mean+s.e. of two experiments. (F) Hypothetical model for the distribution of LAMP‐2A in microdomains at the lysosomal membrane and its changes with CMA activity (see text for details).
Regulation of neuronal commitment in mouse embryonic stem cells by the Reno1/Bahcc1 locus (2020)
Figure 1

Figure 1. Expression of candidate lncRNAs during neuronal differentiation of mouse ES cells. (A) Immunostaining of ES cells (Oct4), NPCs (day4, Nestin), and neurons (day 8, Tuj1). Scale bar: 200µm. (B) Expression levels of candidate lncRNAs during three stages of differentiation, measured by RNA-seq. Gene models of lncRNAs appear in green, adjacent protein-coding genes appear in grey. Blue depicts transcripts from the '+' strand, orange represents transcripts from the '-' strand. All three RNA-seq tracks were scaled separately to the maximum coverage in each locus. (C) qRT-PCR of Reno1, Cox10as1, and lnc-Nr2f1 in ES cells (top) and following eight days of neuronal differentiation (bottom), following knockdown using two different shRNAs, normalized to sh-NT. Mean ± SEM is shown for 3-6 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test).
Regulation of neuronal commitment in mouse embryonic stem cells by the Reno1/Bahcc1 locus (2020)
Figure 2

Figure 2. Knockdown of Reno1 inhibits neuronal differentiation. (A) Immunostaining using anti-TUJ1 antibody of ES-cell-derived neurons following infection with either non-targeting shRNA (sh-NT), or two different shRNAs targeting Reno1. Scale bar: 200µm. (B) Quantification of cell numbers and neurite lengths of ten images of non-overlapping fields for each shRNA. Mean ± SEM is shown, ** P<0.01 (unpaired two sample t-test). (C) Mean expression levels of genes which were down- or upregulated following knockdown (KD) of Reno1 or lnc-Nr2f1 during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV)1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013). (D) Transcriptional response of cell cycle and neuron-related genes. Heatmaps show log2FC of cells infected with shRNAs targeting Reno1 or lnc-Nr2f1 compared to cells infected with sh-NT, following eight days of neuronal differentiation, and of day eight of differentiation compared to ES cells during differentiation of WT cells.
Regulation of neuronal commitment in mouse embryonic stem cells by the Reno1/Bahcc1 locus (2020)
Figure 3

Figure 3. Reno1 is a deeply conserved lncRNA, found in close spatial proximity to Bahcc1. (A) Reno1 locus in six vertebrate species. Shaded region indicates the Reno1 locus. Blue gene models are from RefSeq or Ensembl, red gene models are based on PLAR transcript reconstructions (Hezroni et al., 2015). RNA-seq datasets from indicated tissues were taken from publicly-available datasets: BodyMap (Li et al, 2017a) (mouse), Reference Epigenome (human), FAANG (sheep), SRP011985 (opossum), SRP016501 (chicken), DRP000627 (coelacanth). RNA-seq data was scaled separately in each species to the highest coverage in the window, except for opossum and chicken, where a custom upper threshold was set as indicated. (B) The mouse Reno1 locus. Expression levels during neuronal differentiation of ES cells, in different cellular fractions of ES cells (data from (Engreitz et al, 2016)) and motor neurons (GSE90913), and in forebrain during different stages of embryonic development (data from the ENCODE project). Each set of tracks was normalized separately. (C) RENO1 locus in human. Expression levels in dorsolateral prefrontal cortex across six age groups, and in different cellular fractions in fetal and adult cells. Each set of tracks was normalized separately. (D) Reno1 smFISH signal (red) and DAPI staining (blue) in WT and Reno1m/m cells at day eight of neuronal differentiation, imaged using 100x objective. Group sizes indicated in Figure EV2G. (E) Expression levels of Reno1 and Bahcc1 in 30 adult and embryonic tissues. Data from the ENCODE project, quantification was done using RSEM (Li & Dewey, 2011). (F) Targeted Chromosome Conformation Capture (4C) using the Reno1 promoter as bait. Top, smoothed trend lines and raw counts of the contact profile in ES cells (red) or differentiated neurons (black); bottom, domainogram showing mean contact per fragment end for a series of window sizes.
Regulation of neuronal commitment in mouse embryonic stem cells by the Reno1/Bahcc1 locus (2020)
Figure 4

Figure 4. Reno1m/m and Bahcc1m/+ ES cells proliferate normally but fail to survive neuronal differentiation. (A) Generation of Reno1m/m and Bahcc1m/+ ES cells using CRISPR-Cas9. Primers used for colony screening marked by arrows. (B) Expression levels of Reno1 (both the spliced and unspliced isoforms) and Bahcc1 following four days of differentiation of the indicated cells. Mean ± SEM is shown for 3-4 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test). (C) Left: Oct4 staining of the indicated cells. Scale bar: 200 µm. Right: Cell count of the indicated ES cells grown in ES medium. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. (D) Left: Nestin staining of the indicated cells following four days of differentiation. Scale bar: 200 µm. Right: Cell count of the indicated cells following four days of differentiation. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. ** P<0.01 (unpaired two sample t-test). (E) Flow cytometry analyses of dead and live cells in the indicated ES cells following four days of differentiation. Y axis: forward scatter; X axis: PI fluorescence intensity.
Regulation of neuronal commitment in mouse embryonic stem cells by the Reno1/Bahcc1 locus (2020)
Figure 5

Figure 5. Transcriptional response to Reno1 depletion and overexpression at day 2 of differentiation. (A). Heatmap showing log2-transformed fold changes of Reno1 depleted cells compared to their respective controls. All genes which were significantly differentially expressed (P<0.05) in any of the treatments are shown. (B) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of Reno1m/m cells compared to WT cells, for genes with significant (P<0.05) change in cells where Reno1 was perturbed with the indicated method. Number of genes in each group is indicated in the x axis. ** P<0.01 (two-sided Wilcoxon rank sum test). (C) Mean expression levels of genes in genes which were significantly (P<0.05) dysregulated by at least two out of three Reno1 perturbations at day 2 in fig. 5A (n= 168 down-regulated and 94 up-regulated), during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV)1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013). (D) Expression levels of Reno1 following Dox addition in WT and Reno1m/m cells. Mean ± SEM is shown for three independent experiments, * P<0.05 (unpaired two sample t-test). (E) Cell count of WT and Reno1m/m cells following four days of differentiation, with or without Dox addition. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. * P<0.05 (unpaired two sample t-test). (F-G) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of WT (F) or Reno1m/m (G) cell treated with Dox for the first two days of differentiation compared to untreated cells, in genes which were significantly (P<0.05) downregulated (n=252) or upregulated (n=223) by at least two out of three Reno1 perturbations at day 2 in fig. 5A, compared to all genes (n=16,511). ** P<0.01 (two-sided Wilcoxon rank sum test).
Regulation of neuronal commitment in mouse embryonic stem cells by the Reno1/Bahcc1 locus (2020)
Figure 6

Figure 6. Global reduction in chromatin accessibility of H3K4me3-marked regions in Reno1- or Bahcc1-deficient cells (A) Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ratios of ATAC-seq signal for the indicated group of peaks, comparing the indicated genotypes or treatments. Core promoter peaks are <300 nt from a TSS; Extended promoter are more than 300 nt but <2 Kb for a TSS; Gene body peaks overlap transcription units, and Intergenic do not. Each group of peaks was compared to the intergenic peaks. In addition, for each group of peaks, the peaks within 1 Mb of Reno1 where compared to peaks further away. P-values computed using two-sided Wilcoxon rank sum test, and shown only for cases where P<0.05. Number of data points (from left to right): 39; 10,777; 11; 3,550; 17; 14,380; 18; 20,930. (B) Changes in ATAC-seq signal for peaks assigned as falling within core or extended promoters, of genes in the indicated group (up/down-regulated by at least 30% and with adjusted P<0.05) in cells with Reno1 (left) or Bahcc1 (right) KD. P-values computed using two sided Wilcoxon rank sum test. (C) Top: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of changes in ATAC-seq signal on day 2 of neuronal differentiation of peaks from indicated Reno1 perturbation, classified according to the underlying chromatin state in mouse ES cells; Bottom: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ChIP-seq coverage of the indicated chromatin marks in ENCODE mouse ES data in the peak regions. Number of peaks in each group is indicated at the bottom. (D) Correlation between changes in chromatin accessibility on day 2 of neuronal differentiation in the indicated perturbations and H3K4me3 or Fam60a ChIP coverage from mouse ES cells (H3K4me3 is from ENCODE project and Fam60a from (Streubel et al., 2017)). Coefficient and P-value computed using Spearman's correlation. n=49,722 peaks.

Expanded View Figure Legends
BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth (2020)
Figure 1

Figure 1. BIC1 was identified as a BZR1-interacting protein.

A Luciferase complementation imaging (LCI) assays showing that BIC1 interacts with BZR1. nLUC-BZR1 was co-transformed with cLUC-CCA1, cLUC-TOC1, cLUC-ELF3, cLUC-LUX, cLUC-BIC1, cLUC, respectively, in leaf epidermal cells of Nicotiana benthamiana as indicated.

B BiFC assays showing the interaction of BIC1 and BZR1 mainly in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-BZR1 and cYFP-BIC1. BF, bright field. Scale bars represent 20 μm.

C In vitro pull-down assays showing BIC1 directly interacts with BZR1. Purified GST-BIC1 proteins were incubated with MBP or MBP-BZR1 for the MBP pull-down assay. Arrowhead indicates specific bands.

D Co-immunoprecipitation (Co-IP) assays showing the interaction between BIC1 and BZR1 in vivo. Seedlings were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively. * indicate specific bands. The cross-reacting lower band on IP blot is the heavy chain after IP.
BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth (2020)
Figure 2

Figure 2. BIC1 positively regulates BR responses.

A, B Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment. Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 seedlings were grown for 6 days on 1/2 MS medium supplemented with 1 μM brassinazole (BRZ) plus different concentrations of epibrassinolide (eBL). Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD; n>20. Scale bars, 2 mm.

C RT-qPCR analysis of BZR1-activated gene expression in the bic1bic2 double mutants. The 6-d-old Col-0 and bic1bic2 seedlings grown on 1/2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL (+eBL) or not (-eBL) for 1 hour. The PP2A gene served as internal control. Data are means ± SD (n=3).

D Heat map of BIC1-regulated genes. The Col-0 and 35S:BIC1-YFP seedlings were grown on 1/2 MS medium for 10 days. Three biologic replicates were performed. The colored bar beneath the map indicates fold change (log2 value).

E BIC1 facilitates the expression of cell elongation genes. RT-qPCR analysis of genes selected in transcriptomic data. The Col-0, 35S:BIC1-YFP and 35S:BIC1-Flag seedlings were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3).

F Gene Ontology analyses of BIC1-regulated genes. Numbers indicate -lg p Value.

G The Venn diagram shows significant overlap between BIC1-regulated and BZR1-target and PIF4-target genes.

H Distribution of BIC1 activated or repressed genes among the overlap genes in (G).
BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth (2020)
Figure 3

Figure 3. BIC1 and BZR1 synergistically promote hypocotyl elongation.

A, B BIC1 and BZR1 synergistically promote hypocotyl elongation. The Col-0, 35S:BZR1-MYC, 35S:BIC1-YFP and 35S:BZR1-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.

C-E BR signaling is enhanced in the 35S:BZR1-MYC/35S:BIC1-YFP plants. Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole (BRZ) plus a gradient of concentrations of epibrassinolide (eBL) under long-day conditions. Images of the representative seedlings when grown with 500 nM eBL (+eBL) or not (-eBL) are shown in (C), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (D) and (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.

F, G BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (F), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (G). Data are means ± SD (n>20). **p < 0.01, n.s. indicates no significant difference, (Student's t-test). Scale bar, 2 mm.

H BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. The Col-0, 35S:BZR1-MYC, bic1bic2, 35S:BZR1-MYC/bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days and then the hypocotyl lengths were measured. Data are means ± SD (n>20).
BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth (2020)
Figure 4

Figure 4. BR-stabilized BIC1 has transcriptional activation activity.

A BR enhances the stability of BIC1 proteins. The 5-d-old 35S:BIC1-YFP transgenic plants were treated with 100 μM cycloheximide (CHX) or co-treated with 100 μM CHX and 1 μM epibrassinolide (eBL). Samples were collected at indicated time points and BIC1-YFP protein levels were analyzed by western blots using anti-GFP antibody. Actin was used to verify equal protein loadings. Intensity of the bands was measured using Adobe Photoshop CS3 Extended program. Three independent experiments were performed with similar results.

B Scheme represents full length and truncated versions of BIC1. NT, amino terminal domain; CT, carboxyl terminal domain; CID, CRY interacting domain.

C Transactivation analysis of BIC1 using a yeast assays. The GAL4 DNA-binding domain (BD) alone was used as the negative control. SD-W, synthetic dextrose medium lacking Trp; SD-WH, synthetic dextrose medium lacking both Trp and His.

D Constructs used for BIC1 transcriptional activity assays as shown in (E). VP16 was used as a positive control. TATA, TATA box for DNA binding; LUC, firefly luciferase; REN, Renilla luciferase; NOS, Nopaline Synthase terminator.

E Transient transcriptional activity analysis in Arabidopsis protoplasts illustrating the transcriptional activation activity of BIC1. The relative luciferase activities were calculated by normalizing the LUC values against REN. Error bars represent SD (n = 3). **p < 0.01, as determined by a Student's t-test.

F Constructs used for BIC1 transcriptional activity assay as shown in (G).

G Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1 synergistically activate PRE5 promoter. The ProPRE5:LUC reporter was co-transformed with the indicated effector constructs. The LUC/REN ratio represents the ProPRE5:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3).

H, I BIC1-mediated hypocotyl elongation was reduced by the addition of SRDX motif. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (H), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (I). #1, #2, #3 represent different 35S:BIC1-SRDX-Flag transgenic lines. Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.
BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth (2020)
Figure 5

Figure 5. BIC1 physically and genetically interacts with PIF4.

LCI assays showing that BIC1 interacts with PIF4. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nLUC-BIC1 or nLUC and cLUC-PIF4 or cLUC as indicated.

B In vitro pull-down assays showing that BIC1 directly interacts with PIF4. Purified GST-BIC1 proteins were incubated with MBP or MBP-PIF4 for the MBP pull-down assay. Arrowhead indicates specific bands.

C Co-IP assays showing the interaction between BIC1 and PIF4 in vivo. The 35S:BIC1-YFP, 35S:PIF4-MYC and 35S:PIF4-MYC/35S:BIC1-YFP transgenic plants were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively.

D, E Phenotypic analyses showing that BIC1 and PIF4 synergistically promote hypocotyl elongation. The Col-0, 35S:BIC1-YFP, 35S:PIF4-MYC and 35S:PIF4-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (D), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.

F RT-qPCR analysis showing that BIC1 and PIF4 synergistically activate PIF4 target genes. The seedlings growth conditions were same as (D). The PP2A gene was used as an internal control. Data are means ± SD (n=3).

G,H Phenotypic analyses showing that PIF4-induced hypocotyl elongation is partially. dependent on the function of BIC1 and BIC2. The Col-0, bic1bic2, PIF4-MYC and PIF4-MYC/bic1bic2 plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (G) and the hypocotyl lengths of indicated genotypes were measured and are shown in (H). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.

I RT-qPCR analysis showing that PIF4-induced expression of target genes is partially dependent the function of BIC1 and BIC2. The seedlings growth conditions were same as (G). The PP2A gene was analyzed as an internal control. Data are means ± SD (n=3). **p < 0.01, as determined by Student's t-test.

J, K Phenotypic analyses showing that BIC1-induced hypocotyl elongation is largely dependent on the function of PIFs. The Col-0, pifQ, 35S:BIC1-Flag, 35S:BIC1-flag/pifQ #7 and BIC1-flag/pifQ #8 plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (J) and the hypocotyl lengths of indicated genotypes were measured and are shown in (K). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.