EMBO/soda-nel huggingface dataset:
A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP modulated NFAT signaling (2021)
Figure 1

Figure 1. Identification of a novel STIM1 splice variant.

A. Schematic exon structure of Stim1 gene with conventional exons depicted in grey. Highlighted in red is the alternative exon 11 (named A), and additional mutually exclusive splicing of exon 13 (named L, green) or exon 13 (named B, blue). Alternative usage of 5'UTR regions are indicated with alt1 and alt2. Arrows indicate schematic location of primers, sequences listed in table EV2).

B. Schematic representation of primer annealing sites on the coding mRNA.

C Analytic PCR with primers flanking the splice site using cDNA's of tissues or cells as indicated. L denotes exon L of STIM1L, A denotes additional exon A.

D. qRT-PCR of exonA specific reaction normalized to sum of exon specific and wt specific (total) Stim1 (± SD) in tissues using cDNA derived from 3-4 mice.

E. Schematic protein structure of STIM1 displaying functional motives. Domain A (red) is located 8 amino acids downstream of the inhibitory domain (ID, yellow) after aa 491.

F. Evolutional conservation of domain A. Identical amino acids within black boxes.

G. Western blot showing Stim1 and Stim1A following heterologous expression in HEK293DKO (left); right panel: quantification from 4 transfections, mean ± SD.

H. Endogenous levels of Stim1 in testes from C57BL/6 wt and splice deficient 10A mice, using an antibody detecting the N-term of STIM1, detecting STIM1A (A, red arrow), STIM1 and its glycosylated (wt) and unglycosylated (wt gl.) form.
A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP modulated NFAT signaling (2021)
Figure 2

Figure 2. Stim1A reduces SOCE

A. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar in MEF Stim1/Stim2-/- cells transfected with Stim1- (black trace, n=111), Stim1A (red trace, n=116), the combination Stim1 with Stim1A (blue trace, n=86) or with vector only (grey trace, n=79).

B. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in A. *** p<0.001, Kruskal-Wallis Anova.

C. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in HEK293 cells co-transfected with Orai1 and either Stim1- (black trace, n=119) or Stim1A IRES-mCherry (red trace, n=118).

D. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in C. ** p<0.01 *** p<0.001 Mann-Whitney test.

E. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in primary astrocytes after transfection with non-silencing (siCTL, black trace, n=216) or exonA-specific siRNA (siS1A, red trace, n=263).

F. Efficiency of siRNA as determined by qRTPCR.

G,H. Quantification of parameters shown in (E).*p<0.05, ** p<0.01, Mann-Whitney test.

I. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in primary astrocytes from C57BL/6 wt (CTRL,black trace, n=49) and splice deficient C57BL/6 10ACon mice (10ACon, red trace, n=82).

J. Quantification of parameters determined in [I]. *** p<0.001 Mann-Whitney test.

Data information: Data (except F) were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.
A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP modulated NFAT signaling (2021)
Figure 3

Figure 3. Co-localization and interaction analysis.

A+B. Images showing representative HEK S1/S2-/- cells co expressing YFP-STIM1 (green) or either STIM1-mCherry or STIM1A-mCherry (red) and merged images before (A) and after (B) stimulation with TG.

C. Images showing representative HEK293 cells expressing Orai1-GFP (green = donor molecule) or either STIM1-mCherry (upper panel = acceptor molecule) or STIM1A-mCherry (lower panel = acceptor molecule) after stimulation with TG. Also shown is the overlay of the two channels, as well as the FRET signal.

D. Co-localization analysis of cells from (C). For each condition 9 cells were analyzed using Mander's overlap coefficients (M1 and M2) S1 M1=0.68, M2=0.71; S1A M1=0.55, M2=0.65.

E. Quantification of FRET signal measured in C (STIM1: 0.28, n=37, STIM1A: 0.26, n=39).

F. Distribution of cluster sizes in HEK293 cells transfected with STIM1-mCherry (black, n = 2644) or STIM1A-mCherry (red, n = 4754) at 0.2 µm2 intervals in %.

G. Violin blot of the cluster shown in (F), shown as median, first and third quartile. ns: not significant with Mann Whitney test.

Data information: Data was obtained from three independent transfections and, in D and E, is shown as single values with corresponding median. Scale bars, 10 µm.
A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP modulated NFAT signaling (2021)
Figure 4

Figure 4. Mutation of D503 rescues phenotype of domain A.

A. Amino acids of exon A. Positive charged amino acids are marked blue, negative charged ones are shown in red. Mutated regions are in bold and different mutant combinations investigated in Fig. EV5 are depicted below.

B. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKO1. STIM1 (black, n=145), STIM1A (red, n=157) and STIM1A_D503A (n=185).

C. Relative fluorescence intensities of mCherry-tagged constructs measured in (B).

D. Quantification of changes in resting ratio, TG peak (∆ ratio) and SOCE parameters measured in B.

E. Average traces showing whole-cell current density (CD) over time extracted at -80 mV in HEKO1 cells transfected with STIM1 (black), STIM1A (red) or STIM1A_D503A (blue).

F. Average maximum CDs recorded from cells measured in E (n within bars).

G. Average current-voltage (I- V) relationship of all cells recorded in E.

Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters.
A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP modulated NFAT signaling (2021)
Figure 5

Figure 5. Mutation within the ORAI1 ETON region masks the STIM1A phenotype.

A. Exemplary normalized current responses a -120mV pulse showing fast Ca2+ dependent inactivation (FCDI).

B. Percentage FCDI (plateau/peak, mean±s.e.m.), for HEKS1/S2-/- cells transfected with STIM1+ORAI1 (O1 S1), 22 patch-clamped cells or transfected with STIM1A and ORAI1 (O1 S1A,17 cells) from three independent transfections.

C. Docking model of the STIM1 CAD domain to ORAI at the beginning (red) and at the end (green) of a 50ns MD simulation.

D. close up of interacting residues.

E. alternate docking approach.

F. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKS1/S2-/-.

G. Quantification of the relative reduction of ratio/time or ratio for cells (135<n<202) measured in (F).

H,I. Average traces (mean±s.e.m.) showing whole- cell current density (CD) over time extracted at -80 mV in in HEKS1/S2-/- cells co-transfected either with STIM1 (black) or STIM1A (red) and ORAI1 (H) or with ORAI1 R77E (I) and recorded using extracellular solution containing 2 mM Ca2+.

J. Average maximum CDs recorded from cells measured in [H,I] (n within bars).

Data information: In G, ***P<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test; in B,J, *P<0.05, **P<0.01, unpaired T test with Welch's correction. Data points (total n) were obtained from three biological replicates with each three technical replicates (with multiple cells) and shown as mean±s.e.m for traces (F) and as scatter plots with the underlying boxes showing the means for individual parameters (G). For patch-clamp experiments, number in bars (B,J) indicate measured cells, which were from three independent transfections.
A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP modulated NFAT signaling (2021)
Figure 6

Figure 6. Domain A interferes with an interaction of STIM1 with PDE8B and increases NFAT translocation

A. Mean LFQ intensities of interaction candidates of three replicates each of pull-down experiments of HA-tagged STIM1A-mCherry and STIM1-mCherry after expression and TG activation in HEKS1/S2-/-cells. Eluates were analyzed via mass spectrometry and only hits with significant differential scores (p-values <0.05, Student T-test) are shown.

B. Hits from (A) tested for interaction with commercially available constructs quantified with bimolecular fluorescence complementation (BiFC) via flow cytometry. HEKS1/S2-/- were transfected with STIM1-YFPC (black) or STIM1A-YFPC (red) in combination with POI-YFPN and screened for YFP+ cells via FACS, results (mean ± SD) from three transfections with each 10.000 sorted cells.

C. Correlation of the mean LFQ intensities from (A) and YFP+ cells from BiFC assay from (B). Dotted lines show differences in interaction of the POIs with STIM1 in comparison with STIM1A.

D. Ratio nuclear NFAT-GFP vs. cytosolic GFP intensity normalized to t=0 (mean±s.e.m.) after transfection of SH-SY5Y S1-/- cells with STIM (n=42), STIM1A (n=82), STIM1A_D503A (n=49) or vector only (ØSTIM, n=15) IRES mCherry and induction of SOCE after stimulation with 1 µM TG. Data (total n as indicated) from at least three independent transfections.

E. Normalized endpoint ratios at 30 min (mean±s.e.m) from numbers indicated in (D). * p< 0.05, Kruskal-Wallis Anova with Dunn's multiple comparisons test.
A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP modulated NFAT signaling (2021)
Figure 7

Figure 7. STIM1A alters PDE8B- and cAMP dependent NFAT translocation.

A. Time course of translocation (Nuclear NFAT-GFP signal/cytosolic signal normalized to TG addition at t=0, mean±s.e.m, for each time point) after transfection of indicated constructs and preincubation with 1 µM PF-04957325 (analyzed cells: STIM1 + PF n = 75, STIM1A + PF n = 50, ØSTIM + PF n = 9) Data (total n as indicated) from at least three independent transfections. * p< 0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test.

B. PF-04957325 induced difference of the nuclear NFAT-GFP signal vs. cytosolic signal from (A) to mean of control without the blocker (see Fig. 6D). * p< 0.05, **p<0.01, ***p<0.001,Mann-Whitney test of differences..

C. Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment, shown as mean±s.e.m , ØSTIM + 6Bnz-cAMP/8-pCPT-cAMP n = 15/14 cells; STIM1 + 6Bnz-cAMP/8-pCPT-cAMP: n = 87/66, STIM1A + 6Bnz-cAMP : n = 78/67; from three independent transfections.

D. Traces showing average changes (mean) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of TG as indicated in the upper bar in SH-SY5Y S1-/- cells transfected with STIM1,STIMA or STIM1A_D503A IRES mCherry and measured with and without incubation with 1 µM PF-04957325 (n=82-134).

E. Area under the curve calculated from individual cells as measured in (D). Bars indicate mean±s.e.m.

F. schematic representation of the RflincA, modified after (Ohta et al., 2018).

G. Normalized fluorescence signal change (mean±s.e.m) of R-FlincA, SH-SY5Y S1-/- cells expressing STIM1 (n=20) or STIM1A (n=22) IRES GFP variants and the indicator were stimulated by TG (1 μM). To avoid photobleaching, values were only recorded after 5 and 30 min, respectively The mutant R-Flinc (n=15) was measured in SH-SY5Y wt cells after TG stimulation.

Data information: *p<0.05, **p<0.01, ***p<0.001, 2way Anova in (B), Kruskal-Wallis Anova with Dunn's multiple comparison test (C, E, F, G. Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.
Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits (2020)
Figure 3

Figure 3.

Pre-miRNAs are trafficked along microtubules associated with vesicles

A. Schematic of the experimental paradigm. Concentrations used: 5µM MB; 0.5µg/µL pCS2-CD63-eGFP. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.

B. Representative axon where MB-labeled pre-miR-181a-1 (red) and CD63-eGFP-labeled vesicles (green) are co-trafficked (white arrows). Abbreviations: MB, molecular beacon. Scale bars: 5µm.

C. Representative kymographs. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon. Scale bars: 5µm.

D. Composite kymograph shown in (C) where the individual traces where drawn and color coded. Yellow trajectories represent co-trafficking MB-labeled pre-miRNA (red) and CD63-eGFP-labeled vesicle (green). Abbreviations: MB, molecular beacon. Scale bars: 5µm.

E. Representative time-lapse depicting MB-labeled pre-miR-181a-1 (red arrow) and CD63-eGFP-positive vesicle (green arrow) co-trafficked along the axon shaft to the growth cone (delineated with dashed white lines) wrist (white arrowhead) and central domain (white star). Scale bars: 5µm.

F. Frequency (in percentage) of MB-labeled pre-miR-181a-1 co-trafficked with CD63-eGFP-positive vesicles. Each data point corresponds to one axon. Total number of puncta and axons analyzed: 253 puncta, 22 axons (MB+), 306 puncta, 22 axons (CD63+). n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.

G. Frequency distribution (in percentage) of MB-labeled pre-miR-181a-1 co-trafficked with CD63-eGFP-positive vesicles. Each data point corresponds to one axon. Total number of puncta and axons analyzed: 92 puncta, 22 axons (anterograde), 78 puncta, 22 axons (retrograde). n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.

H. Schematic of the experimental paradigm. Concentrations used: 250ng/µL cy3-pre-miR-181a-1; 0.5µg/µL pCS2-CD63-eGFP. Abbreviations: CD63, CD63-eGFP.

I. Representative image of an electroporated and dissociated RGC. Abbreviations: CD63, CD63-eGFP. Scale bars: 5µm.

J. Schematic of the analysis. Abbreviations: Pre-miRNA, pre-miRNA-181a-1.

K,L. Fluorescence intensity of MB (K) or CD63-eGFP (L) measured in axons and growth cone central domains. Each data point corresponds to one axon or a growth cone central domain. Total number of axons analyzed: 16. n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon; GC, growth cone.

Data information: *** p<0.001, **** p<0.0001. Data were normally distributed (Shapiro-Wilk test) (G,K). Unpaired t-test (G). Paired t-test (K). Data were not normally distributed (Shapiro-Wilk test). Wilcoxon matched pair test (L).
Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits (2020)
Figure 5

Figure 5.

Dicer and Ago2 are present within axons

A. Schematic representation of the experimental protocol.

B. Representative Xenopus growth cones stained with anti-Dicer and anti-Ago2. White dashed lines delineate growth cones. Scale bars: 5µm.

C. Representative E13.5 mice brain cross-section (dashed red line in the schematic) comprising the optic nerve (ON) stained with anti-neurofilament and anti-HA antibodies to detect RGC axons and (FLAG-HA2)-Dicer, respectively. Note the absence of HA signal in wild type (WT) mice. The white dashed line delineates the ON. Zoom of the triple stained ON (right panel): Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se (arrowheads). n=3 independent experiments. Abbreviations: WT, wild type; HA, (FLAG-HA2)-Dicer; E13.5, embryonic day 13.5; ONH, optic nerve head; ON, optic nerve. Scale bars: 30µm.

D. Representative P0 superior colliculus stratum griseum superficiale (SGS) (sagittal sectioning of P0 brains along the dashed red line in the schematic, maximal projection of 5 µm depth). Note the presence of Dicer within the SGS marked with neurofilament. n=3 independent experiments. Abbreviations: WT, wild type; P0, postnatal day 0. Scale bars: 30µm.
Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits (2020)
Figure 6

Figure 6.

Pre-miRNAs are processed locally in response to cues and newly generated miRNAs are important for growth cone steering ex vivo

A. Schematic representation of the experimental paradigm. Concentrations used: 200ng/mL Sema3A and Slit2.

B, C. Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Sema3A (B) or Slit2 (C) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=3 (B), n=4 (C) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.

D. Schematic representation of the experimental paradigm. Abbreviations: co-MO, control morpholino.

E,F. Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-5p) (E) or 2µM (MOs-3p) (F). Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.

Data Information: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Data were not normally distributed (Shapiro-Wilk test) (B,C). Two-tailed Mann Whitney test (B,C). Two-way ANOVA followed by Tukey's multiple comparison post-hoc test (E,F).
Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits (2020)
Figure 8

Figure 8.

Newly generated miRNAs silence the locally translated transcript TUBB3

A. Schematic representation of the experimental paradigm. Plasmid concentrations used: 1μg/μL pCS2-mRFP; 0.7μg/μL pCS2+Venus-no 3'UTR or pCS2+Venus-3'UTR of interest. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated.

B-G. Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 100µM cycloheximide; 2µM co-MO or MOs-3p. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants (B, F, G), isolated axons (C) or isolated transfected axons (D) were used for FRAP analysis. In vivo (E), whole embryos with exposed brain were used. The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution. (B) 200ng/mL Sema3A was bath applied with or without 100µM cycloheximide (CHX, a translational blocker). Mutating TUBB3 3'UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons. (E) The red area on the brain schematic indicate Sema3A expressing territories. Numbers of axons analyzed are reported between brackets. n=5 (B), n=4 (C), n=3 (D-G) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; wt, wild type; mut, miR-181-5p responsive elements mutated; CHX, cycloheximide; TUBB3, tubulin beta 3 class III; APP, Amyloid beta precursor protein; THBS1, Thrombospondin 1; co-MO, control morpholino.

H,I. Representative growth cones depicting Venus fluorescence intensity as a heatmap. Dashed white lines delineate the growth cones. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated; CHX, cycloheximide; TUBB3, tubulin beta 3 class III. Scale bars: 10µm.

Data information: Statistics: ** p<0.01, **** p<0.0001. Dashed black lines represent least-square fits to a single-exponential decay equation (B-G). Extra sum-of-squares F test. Exact p-value of all FRAP experiments and row statistics are reported in Appendix Table EV3.
Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy (2012)
Figure 1

Normal skeletal muscle morphology in hibernating squirrels.

A.

Left column, an active summer squirrel; right column, a torpid squirrel.

B.

The morphology of quadriceps is unchanged by hibernation as seen in haematoxylin and eosin (H&E) stained sections (scale bar 90?µm).

C.

Dystrophin staining was performed to outline the sarcolemma to determine percentage distribution of minimum Feret's diameter.

D.

Average?±?SD of minimum Feret's diameter in quadriceps (p?=?0.26) and tibialis anterior (p?=?0.33) muscles is not significantly different between summer and hibernation.
Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy (2012)
Figure 2

Evaluation of protein degradation and synthesis pathways.

A.

Western blot of quadriceps muscle from summer active (S) and hibernating (H) squirrels using antibodies against the proteins indicated. An increased abundance of P-Foxo3a (serine-253) is accompanied by a decrease in P-Akt (serine-478). Corresponding densitometry of P-Akt and P-Foxo3a as a function of total Akt and Foxo3a.

B.

Relative mRNA levels for four Foxo3a downstream targets show no significant changes during hibernation.

C.

Western blots analysis of mTOR downstream targets shows significant upregulation of P-P70S6K and P-4E-BP1 during hibernation.
Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy (2012)
Figure 3

SGK1 in skeletal muscle is critical for preservation of skeletal muscle.

A.

Western blot analyses and densitometry of quadriceps muscles from non-hibernating and hibernating squirrels demonstrates significant increases in total SGK1 and phosphorylated SGK1. Immunostaining of quadriceps muscle for total SGK1 shows upregulation during hibernation.

B.

Levels of phosphorylated Foxo3a in S315 and T32 are significantly increased during hibernation. Densitometric analysis of P-Foxo3a as a function of Foxo3a.
Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy (2012)
Figure 4

Sgk1−/− mice exhibit decreased muscle fiber size and impaired muscle function.

A.

The morphology of tibialis anterior muscle as revealed by haematoxylineosin staining shows increased variation in muscle fiber size with numerous atrophic muscle fibers (scale bar 90?µm).

B.

Percentage distribution and mean minimum Feret's diameter in tibialis anterior muscle (p?=?0.02).

C.

Increased levels of phosphorylated Akt (S478) in skeletal muscles of sgk1-/- mice.

D.

sgk1-/- mice exhibit decreased running distance after 36 days of exposure to a running wheel, (p?=?0.01; n?=?6 animals per group).

E.

Isometric force from soleus muscles of WT and sgk1-/- mice during repetitive electrical stimulations for 500?ms. Amplitudes of isometric force from soleus muscles at two stimulation frequencies. Specific force is plotted in all cases. Mean values?±?SD from n?=?9 (sgk1-/-) and n?=?10 (WT) independent animals (p?=?0.01).