EMBO/soda-nel huggingface dataset:
Lrig1 is a cell-intrinsic modulator of hippocampal dendrite complexity and BDNF signaling (2016)
Figure 1

Figure 1. Developmental expression and somatodendritic localization of Lrig1 in hippocampal neurons

A. Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real-time RT-PCR. The results are shown as mean SEM of n=3 independent assays. The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp (TATA binding protein). The insert shows the expression of Lrig1 in embryonic E17.5 rat hippocampus examined by RT-PCR. Control sample without reverse transcriptase (-RT) is also shown.

B. Localization of Lrig1 (red) in coronal sections from P15 rat brain by immunofluorescence. Anti-Lrig1ECD antibodies label dentate gyrus cells, CA1-CA3 hippocampal neurons and pyramidal cortical neurons (*). Scale bar, 400 m.

C-E. Confocal image of cortical (C) and hippocampal (D-E) pyramidal neurons stained with rabbit anti-Lrig1ECD antibody. Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1-CA3 hippocampal and pyramidal cortical neurons (layer V). Scale bars, 20 m.

F. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the neuronal marker ßIII tubulin (green) in dissociated hippocampal cells cultured for 7 DIV. Yellow indicates neuronal expression of Lrig1. Scale bar, 15 m.

G. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the astrocytic marker S100 (green) in dissociated hippocampal cells cultured for 7 DIV. Scale bar, 20 m.

H. Localization of Lrig1 (red) with anti-Lrig1ECD antibody and the somatodendritic marker MAP-2 (blue) by immunocytochemistry in dissociated rat hippocampal neurons after 12 DIV. Scale bar, 20 m.

All data represent at least three independent experiments.
Lrig1 is a cell-intrinsic modulator of hippocampal dendrite complexity and BDNF signaling (2016)
Figure 2

Figure 2. Lrig1 downregulation potentiates dendritic growth and branching of hippocampal neurons

A. Representative images of mouse hippocampal neurons transfected with either GFP-expressing control or Lrig1 shRNA vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Scale bar, 15 m. Boxed area represents a higher-magnification image showing the profuse proximal dendritic arborization of Lrig1-shRNA transfected neurons.

B. Sholl analysis of the dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Data are shown as mean SEM of n=3 independent experiments. *p<0.05 and **p<0.01 by two-way ANOVA followed by Bonferroni multiple comparisons test.

C-G. Quantification of primary (C), and secondary (D) dendrites as well as total dendritic branching (E), terminal dendritic points (F), and total dendritic length (G) of hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.

H. Knockdown efficiency was analyzed by real-time RT-PCR in MN1 cells transfected with control or Lrig1 shRNA vectors. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs. Data are shown as individual values of a representative assay measured in triplicates. n=2 independent experiments were performed.

I. Representative images of MAP-2 immunostained hippocampal neurons obtained from wild-type and Lrig1-deficient mice cultured for 7 days in vitro (7 DIV). Scale bar, 15 m.

J. Sholl analysis of the dendritic arbor from MAP-2 stained hippocampal neurons (7 DIV) isolated from wild type and Lrig1-deficient mice. Data are shown as mean SEM of n=3 independent experiments. *p<0.05 by two-way ANOVA followed by Bonferroni multiple comparisons test.

K-M. Quantification of the number of primary dendrites (K), secondary dendrites (L) and total dendritic branching (M) from MAP-2 stained hippocampal neurons (7 DIV) isolated from wild-type and Lrig1-deficient mice. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.

Note that the different scales of values obtained between shRNA-mediated knockdown and knockout neurons (panels B and J) are due to differences in the experimental conditions between both assays (densities of the cultures and days that the cells were maintained in culture).
Lrig1 is a cell-intrinsic modulator of hippocampal dendrite complexity and BDNF signaling (2016)
Figure 3

Figure 3. Lrig1 controls apical dendrite arborization of CA1 pyramidal neurons

A. Representative images and drawings of Golgi-stained hippocampal CA1 pyramidal neurons from 4 weeks-old wild type and Lrig1-null littermate mice. Scale bar, 15 m.

B. Quantification of the number of primary dendrites and branching of apical and basal dendritic arbors of hippocampal CA1 pyramidal neurons from 4-5 weeks-old control (wild type/heterozygous) and Lrig1-null littermate mice. The results are shown as mean SEM of independent determinations performed in n=4 mice of each genotype. *p<0.05, **p<0.001 by Students t test. NS, not significant.

C. Cummulative dendrite crossings of concentric circles of increasing radius (10 μm ring interval) centering the reference point at the cell body. These values were obtained by Sholl analysis and represent the summatory of the dendritic crossings registered within the first 60 m closest to the soma. The results are shown as mean SEM of independent determinations performed in n=4 mice of each genotype. *p<0.05 by Students t test.

D. Sholl analysis of apical and basal dendritic arbors of hippocampal CA1 pyramidal neurons from 4-5 weeks-old control (wild type/heterozygous) and Lrig1-null littermate mice. The results are shown as mean SEM. *p<0.05, **p<0.001 by two-way ANOVA followed by Bonferroni multiple comparisons test.

Quantifications showed in B-D were performed in n=60 neurons from 4 wild type/heterozygous mice and 4 Lrig1-null littermate mice (n=4).
Lrig1 is a cell-intrinsic modulator of hippocampal dendrite complexity and BDNF signaling (2016)
Figure 4

Figure 4. Lrig1 knockout mice exhibit social interaction impairments

A. Schematic diagram of the social interaction device indicating the social and the empty chambers.

B, C. Mice were simultaneously exposed to an empty container and a caged unfamiliar juvenile mouse (social enclosure, stranger 1). Lrig1-mutant mice exhibit social interaction defects as determined by the time spent interacting (sniffing) with the stranger enclosure (B) and the percentage of total interaction time with stranger in the three-chamber social interaction test (C). Dashed line in (C) represents chance-level performance (i.e. 50%) when mice equally explore the social enclosure and the empty container. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance between wt and knockout mice are *p<0.05 by Students t test.

D. Schematic diagram of the social novelty test. In the test for social novelty, a second stranger (stranger 2) mouse was introduced in the empty container.

E. In the preference for social novelty task, wt mice showed a preference for social novelty, while mutant mice showed no significant preference for novel target (stranger 2). Mutants also spent significantly less time interacting with the novel mice compared to controls. Data represent means ± SEM of independent determinations performed in n=8-9 mice of each genotype, and the statistical significance are as follow: *p<0.05 and **p<0.005 by ANOVA followed by Student-Neuman Keuls multiple comparison test.
Lrig1 is a cell-intrinsic modulator of hippocampal dendrite complexity and BDNF signaling (2016)
Figure 5

Figure 5. Lrig1 overexpression restricts hippocampal dendrite morphology and dendritic spine density in response to BDNF

A. Schematic representation of Lrig1 mutants are shown on the left. Expression levels of these mutants were analysed in transfected cell extracts by immunoblotting with anti-Flag antibodies.

B. Representative images of rat hippocampal neurons transfected at DIV8 with empty vector, full length (FL) Flag-tagged Lrig1, or Lrig1 mutant lacking the LRR domain (LRR) in combination with an enhanced green fluorescent protein (GFP) expression vector. After transfection at 9 DIV, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h. Then, hippocampal cultures at 11 DIV were fixed and stained with anti-Flag antibodies to control Lrig1 expression. Scale bar, 15 m.

C, D. Quantification of the effects of Flag-Lrig1 constructs on BDNF-induced primary (C) and secondary (D) dendrite formation of hippocampal neurons treated as indicated in A. The results are shown as mean SEM of 3 independent experiments. *p<0.05, **p<0.01 by one-way ANOVA followed by Tukey´s multiple comparison test. NS, not significant.

E. Representative confocal images of dendritic shafts containing spines from hippocampal neurons transfected at 15 DIV with either control vector or Flag-Lrig1 construct together with GFP. After transfection, neurons were cultured in the absence or in the presence of BDNF (30 ng/ml) for 48 h (15+3 DIV). Scale bar, 5 m.

F. Quantification of the effect of Lrig1 overexpression on neurotrophin-induced spine density. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by one-way ANOVA followed by Student-Neuman Keuls multiple comparison test.
Lrig1 is a cell-intrinsic modulator of hippocampal dendrite complexity and BDNF signaling (2016)
Figure 7

Figure 7. Lrig1 downregulation enhances TrkB signaling and dendrite development of primary hippocampal neurons in responses to BDNF.

A. Immunoblot showing TrkB activation in hippocampal neurons cultured from Lrig1 heterozygous (+/-) and Lrig1 knockout (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control.

B. Fold of TrkB activation relative to unstimulated control group (phospho-TrkB at Tyr705) in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).

C. Immunoblot showing MAPK activation in hippocampal neurons cultured from Lrig1 wild-type (+/+) and Lrig1-deficient (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for the indicated times. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control. Fold of MAPK activation relative to tubulin is indicated.

D. Fold of MAPK activation (P-MAPK) relative to untreated control group in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).

E. Representative images of mouse hippocampal neurons transfected at 4 DIV with either GFP-expressing control vector or Lrig1 shRNA-GFP plasmid. After transfection, neurons were cultured in the absence or in the presence of BDNF (25 ng/ml) for 48 h (7 DIV). Arrows indicate branching points along the principal dendrite. Scale bar, 10 m.

F-I. Quantification of primary dendrites (F), secondary dendrites (G), total branching (H) and branchig of the principal dendrite (I) of hippocampal neurons treated as indicated in E. The results are shown as individual values and the means of a representative assay. A total of 30 neurons were analyzed per experimental condition. Similar results were obtained in n=3 independent experiments.

J. Lrig1+/+ and Lrig1-/- hippocampal lysates from postnatal day 11 (P11) mice were immunoblotted against phospho-TrkB (p-TrkB) at Tyr705 and total TrkB. Quantification (mean SD) of the p-TrkB/TrkB ratio between Lrig1+/+ (n=4) and Lrig1-/- (n=3) hippocampi is also shown.
The Sec61 translocon is a therapeutic vulnerability in Multiple Myeloma (2021)
Figure 1

Figure 1 - Sec61 blockade by mycolactone strongly alters the biology and viability of MM cell lines.

A Effect of mycolactone on MM.1S, JIM3 and KMS-11 cell viability after 48 and 72 h of treatment, as measured by exposure of Annexin V and incorporation of PI. Data are Mean % of live, apoptotic and dead cells from technical duplicates, gated as in Appendix Figure S1, relative to total cells.

B, C Inhibitory effect of mycolactone on cell expression of CD38 (Type II) and CD138, IL-6R, and CD40 (Type I) transmembrane proteins (B). Stimulatory effect of mycolactone on cell expression of BCMA, a Type III transmembrane protein (C). Mean Fluorescence Intensities (MFIs) were measured in live cells 48 h after addition of mycolactone. Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments, relative to vehicle-treated controls (Ctrl).

D Inhibitory effect of mycolactone on immunoglobulin (lambda chain) secretion, as quantified by ELISA in culture supernatants after 24 h of treatment. Data are Mean concentrations ± SD of secreted lambda chain (µg.mL-1) from technical duplicates of 2 independent experiment (MM.1S) or Mean of secreted lambda chain concentrations (µg.mL-1) from technical duplicates (JIM3 and KMS-11), relative to vehicle-treated controls.

E Correlation between immunoglobulin lambda chain (Ig) secretion after 24 h (D) and MM cell line mortality after 72 h (A), in the 3 cell lines treated with 50 nM mycolactone. Slopes (R) and statistical significance (p values) are indicated.

Data information: In A (all cell lines) and D (JIM3 and KMS-11), shown data are representative of 2 independent experiments with similar results.
The Sec61 translocon is a therapeutic vulnerability in Multiple Myeloma (2021)
Figure 3

Figure 3 - The mycolactone-BZ combination activates pro-apoptotic UPR in MM.1S cells.

A Diagram illustrating the pro-apoptotic pathways activated by UPR sensors PERK, ATF6 and IRE1 upon accumulation of unfolded proteins in the ER lumen.

B-G In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (B) ATF4 mRNA levels were quantified by qPCR; (C) ATF4 protein levels in cell lysates were assessed by Western blot (top panel) and quantified relatively to GADPH levels (lower panel); (D) CHOP and GADD34 mRNA levels were quantified by qPCR; (E) ATF6 and GAPDH protein levels in cell lysates were assessed by Western blot with molecular weight markers (kDa) indicated on the right; Bands corresponding to glycosylated (G-ATF6), non-glycosylated (NG-ATF6) and cleaved ATF6 are indicated by arrows; (F) Total RNA was isolated and used as a template for RT-PCR of XBP-1 (upper panel) which was then digested with Pst1 and separated on a 2% agarose gel (lower panel) with molecular weight markers (bp) indicated on the left; spliced XBP1 (sXBP1) mRNA levels were also quantified by qPCR and normalized to total (spliced + unspliced) XBP1 mRNA levels, (G) BiP mRNA level were quantified by qPCR.

Data information: Shown mRNA data are Mean fold changes (2-∆∆CT) ± SD, relative to untreated controls (cumulative data of at least 2 independent experiments with technical duplicates or triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6 h), Tunicamycin (Tu, 2 µM, 6 h) or DTT (4 mM, 2 h) were used as positive controls. In C, E and F, shown data are representative of 2 independent experiments with similar results.
The Sec61 translocon is a therapeutic vulnerability in Multiple Myeloma (2021)
Figure 4

Figure 4 - The cytotoxic synergy between mycolactone and BZ is maintained in BZ-resistant cells.

A Parental MM.1S (BzS) and MM.1S BzR cells were incubated with increasing concentrations of BZ for 24 h. The proportion of live (Annexin V- PI-) cells was then measured by flow cytometry as in Appendix Figure S1. Data are Mean % of live cells from technical duplicates, relative to vehicle controls.

B Effect of mycolactone on MM.1S BzR cell viability after 48 and 72 h of treatment, as measured by exposure of Annexin V and incorporation of PI. Data are Mean % of live, apoptotic and dead cells from technical duplicates, gated as in Appendix Figure S1, relative to total cells.

C MM.1S BzR cells were treated for 24 h with mycolactone and/or BZ at the indicated concentrations, or vehicle as control. Data are Mean % ± SD of live, apoptotic and dead cells, gated as in Appendix Figure S1, relative to total cells. N = 6 (cumulative data of 3 independent experiments with technical duplicates).

D Synergy between mycolactone and BZ in the MM.1S BzR lines, when treated as in (C). Data are Mean Loewe scores ± SD, shown as heatmaps. Differences between treated cells and controls by Student's t-test: *p<0.05; N = 6 (cumulative data of 3 independent experiments with technical duplicates).

E ATF4, CHOP, sXBP1 and BiP mRNA levels were quantified by qPCR in MM.1s BzR cells treated as in (C). sXBP1 mRNA levels were normalized to total (spliced + unspliced) XBP1 mRNA level. Data are Mean RNA fold changes (2-∆∆CT) ± SD, relative to untreated controls. N = 5 (cumulative data of 2 independent experiments with technical duplicates and triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.

Data information: In A and B, shown data are representative of 2 independent experiments with similar results.
The Sec61 translocon is a therapeutic vulnerability in Multiple Myeloma (2021)
Figure 5

Figure 5 - The synergy between mycolactone and BZ cytotoxicity extends to mouse B cell lymphomas.

A Effect of mycolactone on v-abl pro-B cell viability after 24 and 48 h of mycolactone treatment, as measured by exposure of Annexin V and incorporation of PI. Data are Mean % of live, apoptotic and dead cells from technical duplicates, gated as in Appendix Figure S1, relative to total cells.

B v-abl pro-B cells were treated for 24 h with mycolactone and/or BZ at the indicated concentrations. The percentage of live, apoptotic and dead cells was then measured by flow cytometry as in Appendix Figure S1. Data are Mean % from technical duplicates, relative to total cells.

C v-abl pro-B cells were treated with mycolactone and/or BZ at the indicated concentrations for 6 h. CHOP mRNA levels were quantified by qPCR. Data are Mean RNA fold changes (2-∆∆CT) ± SD relative to untreated controls. N = 6 (cumulative data of 3 independent experiments with technical duplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated.

D v-abl pro-B cells were treated for 6 h with mycolactone and/or BZ at the indicated concentrations. ATF4 protein levels were assessed in cell lysates by Western blot (top panel) and quantified relative to GADPH levels (lower panel).

Data information: Data shown in A, B and D are representative of experiments performed with two independent v-abl cell clones, with similar results. In C and D, Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.
The Sec61 translocon is a therapeutic vulnerability in Multiple Myeloma (2021)
Figure 6

Figure 6 - MM primary tumors are highly susceptible to mycolactone toxicity and synergy with BZ.

Mononuclear cells from bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients were treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control, for 18 h. Then, MM cells were identified by staining with anti-CD38 and anti-CD138 antibodies using the gating strategy depicted in Appendix Figure S2.

A Following treatment, induction of apoptosis/necrosis was measured by exposure of annexin V and PI incorporation. Data are Mean % of live cells from technical duplicates, relative to controls.

B Synergy between mycolactone and BZ in treated tumors. Data are Mean Loewe scores from technical duplicates, shown as heatmaps.
The Sec61 translocon is a therapeutic vulnerability in Multiple Myeloma (2021)
Figure 7

Figure 7 - Combining mycolactone with BZ delays MM tumor growth in vivo.

A C57BL/6 mice (N=2/group) were injected intraperitoneally with BZ (0.5 mg.Kg-1), BZ (0.5 mg.Kg-1) + mycolactone (0.3 mg.Kg-1), DMSO as vehicle controls every 3.5 days, or left untreated. Blood samples were analyzed 5, 15, 20 and 25 days after the first injection and percentages of CD3+ and CD19+ cells determined by flow cytometry. Results are expressed as % of positive cells among total mononuclear cells, each dot representing the Mean % from 2 mice.

B NSG mice (N=9/group) were injected subcutaneously with 3.106 MM.1S cells at day 0. Seven days later, mice were injected with DMSO, BZ (0.5 mg.Kg-1) and/or mycolactone (0.3 mg.Kg-1) every 3.5 days intraperitoneally and tumor growth was followed by daily measurement of the tumor diameter. Data are Mean tumor diameters ± SD and represent cumulative data from 3 independent experiments with 3 mice/group. Difference between groups were analyzed with Tukey's multiple comparison test using Two-way Anova, with mixed effect model for each time points: *p<0.05; **p<0.01; ***p<0.001 (exact p-values indicated). Only significant differences are shown.

Expanded version figure legends
TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T cell numbers (2020)
Figure 1

Figure 1. Deletion of Tet2 in C57BL/6 mice promotes syngeneic tumor growth.

A Representative image of tumor-bearing mice and tumors after subcutaneous injection of Hepa1-6 hepatoma cells into WT and Tet2-/- (KO) mice.

B Mean tumor volumes in WT and Tet2-/- mice (n=6).

C Tumor weights 18 days after tumor cell injection into WT and KO mice (n=6).

D Kaplan-Meier survival curves of tumor-bearing and tumor-free WT and KO mice (n=15).

E Representative images of lungs with metastasis from WT and KO mice injected with Hepa1-6 cells via tail vein, as well as quantification of the number of lung metastasis (n=4).

F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells, as well as quantification of tumor weights (n=5).

Data information: Data were analyzed with two-tailed Student's t-test (B, C, E, F). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars denote ± SD. Kaplan-Meier curves were used to represent survival, where significance was calculated with the log-rank test (D).