EMBO/soda-nel huggingface dataset:
Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension. (2015)
Figure 7

siRNA knockdown of pulmonary vascular ISCU1/2 in vivo promotes PH

A Schema of the strategy for pharmacologic inhibition of siRNA knockdown of ISCU1/2 in the pulmonary vasculature of mice in vivo by Staramine-mPEG-mediated intravenous delivery.

B, C Pharmacologic inhibition of ISCU (N = 8/group) did not influence miR-210 expression in the lungs either after Norm + SU5415, NS P = 0.8634 (B) or Hyp + SU5416, NS P = 0.9786 (C).

D As compared with control siRNA (siCont) where ISCU1/2 expression was decreased after exposure to Hyp + SU5416 (white bars), pulmonary vascular delivery of siRNA specific for ISCU1/2 (siISCU) via Staramine-mPEG nanocomplexes (Polach et al, 2012) down-regulated ISCU1/2 in < 100-μm pulmonary vessels in Norm + SU5416 and Hyp + SU5416 (N = 5/group, ***P = 0.0006, **P = 0.0019 for Hyp + SU5416, **P = 0.0029 for siCont).

E By electron paramagnetic resonance (EPR) spectroscopy (representative siCont versus siISCU lung), Fe-S cluster signal was decreased (quantitative graph, right) in siISCU mouse lung tissue (N = 4 mice) compared with siCont (N = 5 mice), *P = 0.0143.

F Endothelin-1 was increased in lung tissue after siISCU delivery under normoxic conditions (N = 5/group), **P = 0.0027.

G–I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased pulmonary vascular remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).

J Model of acquired and genetic Fe-S deficiency as a central cause of metabolic dysfunction and PH in mice and humans. Among others, potential translational implications are highlighted in red text.

Data information: Error bars reflect SEM. Mouse tissue scale bar: 50 μm.
Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (2012)
figf1

(a) Outline of the screening strategy. MS, mass spectrometry. See the text for details. (b) Density gradient fractions of NRK cells starved for 8 h were analysed by transmission electron microscopy (upper panel) and blotted for LC3 or LAMP2 (lower panel). Proteins in fraction 1 were identified by mass spectrometric analysis. Three replicates were carried out from three independent purifications. Scale bar, 2 μm. (c) The dark grey circle shows the percentage of proteins identified from all three purifications; the grey circle shows the percentage of proteins identified from two of the three experiments. (d) Diagram of a SAMCell. Each dot represents a self-assembled cell island that can be transfected with an individual RNAi. (e) NRK cells grown on the SAMCell were transfected with FAM-RNAi by reverse transfection and the transfection efficiency was monitored by microscopy. Left panel, FAM-RNAi-positive cells; right panel, differential interference contrast (DIC) image of the cells in the left panel. (f) Functional associations were analysed by STRING. Stronger associations are represented by thicker lines. Uncropped images of blots are shown in Supplementary Fig. S6.
Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (2012)
figf2

(a) NRK cells stably expressing CFP–LC3; LAMP1–YFP were transfected with nonspecific (NS)- or
Cltc (clathrin heavy chain)-RNAi and starved for 12 h. Scale bar, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes after starvation and quantified.
n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Representative TEM micrographs of nonspecific- or Cltc-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm. (d) NRK cell lines stably expressing LAMP1–Cherry; clathrin–GFP were starved for 0 or 4 h and observed by confocal microscopy. As a control, clathrin–GFP-expressing cells were starved for 0 or 4 h and then stained with MitoTracker red. Scale bar, 5 μm. The insets in a, c and d represent enlarged regions of interest. (e) Cells from d were analysed for Pearson’s co-localization coefficent (R(r)).
n = 30 cells from three independent experiments. Error bars indicate the s.d. (f) NRK cells stably expressing LAMP1–Cherry; clathrin–GFP were starved for 4 h and time-lapse images were acquired by spinning-disc microscopy. Scale bar, 500 nm. The white arrows indicate a clathrin punctum moving along a LAMP1-positive structure. (g) NRK cells were starved for 0, 4 or 6 h and then subjected to cell fractionation. Fractions were analysed by western blotting with antibodies against LAMP2 and clathrin heavy chain. 1 is the top fraction. The outline indicates the Lamp2-positive fraction. (h) Cell lines stably expressing clathrin–GFP were starved for 4 h, stained with antibodies against GFP and LAMP1, analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view. (i) Left panel, representative TEM micrographs of purified autolysosomes from starved clathrin–GFP-expressing cells. Scale bar, 250 nm. Right panel, SIM image of purified autolysosomes stained with GFP and LAMP1 from the same experiment. Scale bar, 5 μm. (j) NRK or stable clathrin–GFP-expressing NRK cell lines were analysed by pre-embedding-immuno-electron microscopy using antibodies against GFP. Scale bar, 100 nm. (k) Quantification of the average number of gold particles per image from control or clathrin–GFP cells.
n = 25 images from three independent experiments. The error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.
Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (2012)
figf3

(a) NRK cell lines stably expressing CFP–LC3; LAMP1–YFP were transfected with nonspecific (NS)- or PIP5K1B-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified.
n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Representative TEM micrographs of nonspecific- or PIP5K1B-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm. (d) NRK cells were starved for 4 h, stained with antibodies against PIP5K1B and LAMP1, and then analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view. (e) Fractions from Fig. 2f were analysed by western blotting with an antibody against PIP5K1B. The outline indicates the LAMP2-positive fraction. (f) NRK cells were starved for 4 h, autolysosomes were purified by density fractionation and the quality of purified autolysosomes was monitored by TEM (left panel). Right panel, purified autolysosomes stained with antibodies against PI(4)P and PtdIns(4,5)P2. Scale bar, 500 nm. (g) NRK cells were starved for 0 or 4 h and stained with antibody against PI(4)P. Scale bar, 5 μm. (h) NRK cells lines stably expressing OSBP-PH–GFP;LAMP1–Cherry were starved for 0, 4, or 8 h and observed by confocal microscopy. Scale bars, 5 μm. (i) NRK cell lines stably expressing OSBP-PH–GFP;LAMP1–Cherry were transfected with PIP5K1B-RNAi. At 60 h after transfection, cells were starved for 12 h and analysed by confocal microscopy. Scale bar, 5 μm. (j) NRK cell lines stably expressing LAMP1–Cherry;clathrin–GFP were transfected with nonspecific- or PIP5K1B-RNAi and starved for 12 h, and then observed by confocal microscopy. Scale bars, 5 μm. The insets in a, h–j represent enlarged regions of interest. (k) Cells from g were analysed for Pearson’s co-localization coefficent (R(r)) by Olympus Fluoview.
n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.
Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (2012)
figf4

(a) NRK cells stably expressing CFP–LC3;LAMP1–YFP were transfected with nonspecific (NS)- or PIP5K1A-RNAi and starved for 10 h. The insets represent enlarged regions of interest. Scale bar, 5 μm. (b) Cells from a were assessed for the presence of elongated reformation tubules in a blind fashion after starvation and quantified.
n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Cells from a were assessed for the length of reformation tubules in a blind fashion after starvation and quantified. 50 cells were counted. The average tubule length is indicated above each column. (d) Diagram of reformation tubules.
R, radius of main body;
L, length. (e) Representative image of reformation tubules in nonspecific- or
PIP5K1A-RNAi-transfected cells. The top and bottom panels show two randomly selected images of the respective samples. (f) Cells from a were assessed for the
L/R ratio of reformation tubules in a blind fashion after starvation and quantified. 25 cells were counted. The average
L/R is indicated above each column. (g) Purified reformation tubules were monitored by TEM. Scale bar, 250 nm. (h) Purified reformation tubules stained with antibodies against PI(4)P, PtdIns(4,5)P2 and PIP5K1A. Scale bar, 250 nm. (i) Fractions from Fig. 2f were analysed by western blotting with an antibody against PIP5K1A. The outline indicates the LAMP2-positive fraction. (j) NRK cells stably expressing CFP–LC3;LAMP1–YFP were starved for 8 h, and the budding of proto-lysosomes was monitored by spinning-disc microscopy. Scale bar, 100 nm. (k) NRK cell lines stably expressing LAMP1–Cherry;clathrin–GFP were starved for 8 h and observed by confocal microscopy. The right panels show enlarged regions of interest. Scale bar, 5 μm. (l) NRK cell lines stably expressing LAMP1–Cherry;clathrin–GFP were transfected with nonspecific- or PIP5K1A-RNAi and starved for 8 h. Scale bar, 5 μm. The middle panels show enlarged regions of interest from the upper panels. The distance between clathrin dots on each reformation tubule in the upper panels is shown in diagrammatic form in the lower panels. Black line: reformation tubules; red dot: clathrin. (m) The distance between two clathrin dots on reformation tubules (RTs) in cells from l was accessed in a blind fashion. Twenty-five cells were counted. The average distance between two clathrin dots is indicated above each column. Uncropped images of blots are shown in Supplementary Fig. S6.
Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (2012)
figf5

(a) NRK cells stably expressing CFP–LC3; LAMP1–YFP were transfected with nonspecific (NS)- or AP2-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified.
n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Representative TEM micrographs of nonspecific- or AP2-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm. (d) NRK cells were starved for 4 h, stained with antibodies against AP2 and LAMP1, and then analysed by 3D-SIM. Scale bar, 5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view. (e) Fractions from Fig. 2f were analysed by western blotting with an antibody against AP2α. The outline indicates the LAMP2-positive fraction. (f) Outline of the AP2 recruitment assay. (g) Purified autolysosomes were treated with trypsin, incubated with rat brain cytosol, washed and then analysed by western blot with antibodies against AP2α and LAMP2. (h) Stripped autolysosomes were pretreated with GFP or PtdIns(4,5)P2 antibody and then incubated with rat brain cytosol. Autolysosomes were washed and then analysed by western blotting with antibodies against AP2α and LAMP2. (i) NRK cell lines stably expressing LAMP1–Cherry;clathrin–GFP were transfected with nonspecific- or AP2-RNAi and starved for 12 h. Scale bar, 5 μm. The insets in a, c and i represent enlarged regions of interest. (j) Cells from i were analysed for the Pearson’s co-localization coefficent (R(r)) by Olympus Fluoview.
n = 30 cells from three independent experiments). Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.
Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (2012)
figf6

(a) NRK cells stably expressing CFP–LC3; LAMP1–YFP were transfected with nonspecific (NS)- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified.
n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Representative TEM micrographs of nonspecific- or AP4-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm. (d) NRK cells were starved for 0, 4, 8 or 12 h, and isolated lysosomes/autolysosome were analysed by western blotting using antibodies against AP4 or LAMP2. (e) Purified reformation tubules were stained with antibodies against AP4 and LAMP1. Scale bar, 2 μm. The bottom row shows enlarged regions of interest from the top panel. (f) NRK cells were transfected with GFP and LAMP1–GFP. At 20 h after transfection, cells were starved for 0 or 8 h, and then collected and analysed by immunoprecipitation (IP) and western blotting (WB) of proteins as indicated, using stably expressed LAMP1–GFP as bait. TCL, total cell lysate. The insets in a, c and g represent enlarged regions of interest. (g) NRK cell lines stably expressing LAMP1–Cherry; clathrin–GFP were transfected with nonspecific- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (h) Cells from g were analysed for the Pearson’s co-localization coefficent (R(r)) by Olympus Fluoview.
n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.
Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (2012)
figf7

(a) Purified autolysosomes were treated with trypsin. The trypsin-treated (stripped) autolysosomes were incubated with rat brain cytosol and reaction mixture, and then analysed by western blotting. HC, heavy chain. (b) Scanning electron micrographs of purified autolysosomes, rat brain cytosol, trypsin-treated purified autolysosomes and trypsin-treated purified autolysosomes after the membrane budding assay. Scale bar, 250 nm. The arrows indicate buds. (c) Trypsin-treated autolysosomes were incubated with rat brain cytosol and reaction mixture in the presence of antibodies against GFP, clathrin, AP2, AP3, AP4 and PtdIns(4,5)P2, and then analysed by western blotting. (d) Reactions from c were visualized by scanning electron microscopy. Scale bar, 250 nm. Arrow indicates bud. (e) Samples from d were quantified for the number of buds on each autolysosome. Error bars represent s.d.
n = 50 autolysosomes from three independent experiments. Uncropped images of blots are shown in Supplementary Fig. S6.
Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation (2012)
figf8

(a) NRK cells were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 0, 4, 8 or 12 h. Scale bar, 5 mm. Cells were stained with antibodies against LC3. (b) Cells from a were assessed for LC3 puncta in a blind fashion after starvation and quantified.
n = 100 cells from three independent experiments. Error bars indicate the s.d.
The long non-coding RNA LINC00941 and SPRR5 are novel regulators of human epidermal homeostasis (2018)
Figure 1

Figure 1: LINC00941 acts as a negative regulator of keratinocyte differentiation
A qRT-PCR analysis of LINC00941 repression during keratinocyte differentiation in primary keratinocyte cultures from three different donors compared to undifferentiated keratinocytes (D0) (n = 6 biological replicates). Data is presented as mean ± SD.

B-C siPool mediated LINC00941 knockdown (B) in calcium-induced keratinocyte differentiation, results in increased abundance of early and late differentiation marker transcripts on day 3 of differentiation (C) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).

D-E LINC00941 knockdown in day 3 (D3) organotypic epidermal tissue cultures (D) results in increased expression of key differentiation genes (E) (n = 3-4 tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).

F Immunofluorescence analysis shows increased levels of early and late differentiation proteins. Collagen VII (col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins Keratin 10 and Loricrin are shown in red (n = 3-4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.
The long non-coding RNA LINC00941 and SPRR5 are novel regulators of human epidermal homeostasis (2018)
Figure 2

Figure 2: LINC00941 is a suppressor of keratinocyte differentiation
A Workflow of the performed RNA-sequencing experiment.

B LINC00941 knockdown efficiency (siLINC00941) in epidermal tissue on day 3 of differentiation as obtained by qRT-PCR measurements (n = 4-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).

C Heatmap of differentially expressed genes (padj < 0.05 and -1 > log2FC > 1) upon LINC00941 depletion on day 3 in organotypic epidermal tissue with marked keratinocyte differentiation genes (n = 4-5 epidermal tissue cultures/knockdown group).

D GO term analysis of downregulated (padj < 0.05 and -1 > log2FC <1) genes in LINC00941 deficient organotypic epidermal tissue on day 3 (n = 4-5 epidermal tissue cultures/knockdown group).
The long non-coding RNA LINC00941 and SPRR5 are novel regulators of human epidermal homeostasis (2018)
Figure 3

Figure 3: LINC00941 and p63 control SPRR5, which is induced during differentiation and different from other human SPRRs
A Differentially expressed genes in day 3 LINC00941 depleted epidermal tissue cluster within the epidermal differentiation complex (EDC) (n = 4-5 biological replicates/knockdown group).

B LINC00941 knockdown on day 3 in calcium-induced keratinocyte differentiation cultures results in increased expression of SPRR5 (FC = fold change) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).

C Detected fragments per million (FPM) for SPRR5 in control (siCtrl) as well as LINC00941 deficient (siLINC00941) epidermal tissue for all biological replicates on day 3 (D3) of differentiation as obtained by DeSeq2 (FC = fold change) (n = 4‑5 epidermal tissue cultures/knockdown group).

D-E SPRR5 induction during the course of keratinocyte (KC) differentiation is shown by northern blot (D) as well as by qRT-PCR analysis (E) for four different primary keratinocyte isolates and compared to undifferentiated keratinocytes (n = 8 biological replicates). Data is presented as mean ± SD.

F p63 knockdown on day 3 of keratinocyte differentiation leads to decreased SPRR5 transcript levels (FC = fold change) (n = 3-4 biological replicates /knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).
The long non-coding RNA LINC00941 and SPRR5 are novel regulators of human epidermal homeostasis (2018)
Figure 4

Figure 4: SPRR5 is indispensable for proper keratinocyte differentiation
A-C siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation (A) leads to decreased expression of key differentiation markers as shown by qRT-PCR (B) (FC = fold change) as well as western blot analysis (C) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).

D SPRR5 knockdown efficiency in day 3 (D3) organotypic epidermal tissue (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).

E SPRR5 depletion in regenerated organotypic epidermal tissue results in drastically reduced expression of differentiation proteins as shown by immunofluorescence analysis. Collagen VII (Col VII, green) stain indicates the basement membrane, nuclei are shown in blue and the differentiation proteins filaggrin and loricrin are shown in red (n = 4 tissue cultures/knockdown group; one exemplary picture for each group is depicted). Scale bar: 50 µm.

F SPRR5 knockdown in regenerated epidermal tissue results in reduced transcript levels of key differentiation genes as obtained by qRT-PCR (n = 4 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).
The long non-coding RNA LINC00941 and SPRR5 are novel regulators of human epidermal homeostasis (2018)
Figure 5

Figure 5: SPRR5 controls the keratinocyte differentiation program on a global level
A Workflow of the performed RNA-sequencing experiment (DE = differentially expressed, log2FC = log2(fold change), padj = adjusted p-value).

B Overview of SPRR5 levels in control (siCtrl) and SPRR5 deficient (siSPRR5) epidermal tissue on day 3 and day 4 as obtained by qRT-PCR analysis (n = 3-5 epidermal tissue cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).

C-D Heatmaps of differentially expressed genes (padj < 0.05 and -0.5 > log2FC > 0.5) upon SPRR5 depletion on day 3 (C) or day 4 (D) in organotypic epidermal tissue (n = 3-5 epidermal tissue cultures/knockdown group).

E-F GO term analysis of downregulated (padj < 0.05 and -0.5 > log2FC) genes in SPRR5 deficient organotypic epidermal tissue on day 3 (E) and day 4 (F) (n = 3-5 epidermal tissue cultures/knockdown group).

G Venn diagram of genes that are upregulated (padj < 0.05 and log2FC > 1) after LINC00941 depletion (red) and downregulated (padj < 0.05 and -1 > log2FC) in SPRR5 deficient (yellow) organotypic epidermal tissue on day 3 (n = 3-5 epidermal tissue cultures/knockdown group).

H GO term analysis of genes conversely (upregulated in LINC00941 deficient and downregulated in SPRR5 deficient samples) regulated by LINC00941 and SPRR5 in day 3 epidermal tissue (n = 3-5 epidermal tissue cultures/knockdown group).

I Phylogenetic analysis of all known human SPRR coding-sequences (cds by means of a maximum likelihood approach. The length of the horizontal bar indicates 0.05 mutations/site. Sequence similarity suggests a common ancestor of all SPRRs, but a distinct composition of SPRR5.

Expanded View Level 2 Figure Legends