EMBO/soda-nel huggingface dataset:
Molecular requirements of the B cell antigen receptor for sensing monovalent antigens (2016)
Figure 6

Figure 6. Stimulation of the B-cell with anti-CD19 enhances its response to monovalent antigen.

A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.

F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.

Addition of reagents to the cells is indicated by arrows. Data are representative of three independent experiments.
Nesprin-2 accumulates at the front of the nucleus during confined cell migration (2020)
Figure 1

Figure 1. Giant nesprins accumulate at the front of the nucleus during migration through narrow constrictions.

A, Schematic illustration of the nuclear envelope, depicting the link between the lamina, SUN proteins, giant nesprins and actin.

B, Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope.

C, Brightfield and fluorescence images of a cell migrating through a 2 µm constriction. Arrows indicate areas of accumulation of nesprin-2 at the front of the nucleus as it is deformed through the constriction. (See also Movie EV1.)

D, Schematic illustrating the 5 time points studied in the analysis: Before nucleus deformation ("Before"), once the front of the nucleus reaches the middle of the constriction ("Front@mid"), once the middle of the nucleus reaches the middle of the constriction ("Middle@middle"), a timepoint halfway between Front@mid and Middle@middle ("between"), and late time point after the deformation ("After").

E, Brightfield and fluorescence images of a cell migrating along a 15 µm control channel.

F, Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel. The average intensity at the front and the back is normalized to the intensity at the sides, demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation. Error bars correspond to the standard deviation. Constriction: n=17 cells over 4 biological replicates, 15 µm channels: n=20 cells over 3 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.

G, GFP-nesprin-2 cells deformed by the pressure gradient in a micropipette aspiration device. Note that the GFP signal does not increase at the tip but increases at sides of the nucleus.
Nesprin-2 accumulates at the front of the nucleus during confined cell migration (2020)
Figure 2

Figure 2. The accumulation of nesprins does not associate with lamin accumulation.

A, CRISPR-modified cells (Brightfield, left) that express GFP-nesprin-2 (middle) and mCh-LAC (right) show nesprin (orange arrow) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction. (See also Movie EV 2.)

B, Quantification of intensity of GFP-nesprin-2 and mCh-LAC, normalized to the intensity at the sides. Error bars correspond to the standard deviation. Constriction: n=19 cells over 5 biological replicates, 15 µm: n= 27 over 6 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.
Nesprin-2 accumulates at the front of the nucleus during confined cell migration (2020)
Figure 4

Figure 4. Nesprin accumulation is due to the actin cytoskeleton.

A, Expression of the artificial mini-nesprin-2G construct results in significant nesprin accumulation (orange arrow) in cells migrating through narrow constrictions. (See also Movie EV4.)

B, Schematic depicting the endogenous protein, and the artificial mini-nesprin-2G and MUT-mini-nesprin-2G constructs. Both constructs contain the first two and last two spectrin repeats of the endogenous protein; the mutant construct presents two mutations on the actin-binding domain that prevents it from binding to actin.

C, Quantification of the mini-nesprin-2G accumulation of cells migrating through narrow constrictions. Note that the mutant construct does not accumulate at the front of the nucleus. (See also movie EV4 and movie EV5.) Error bars correspond to the standard deviation. Mini-nesprin-2G: n=10 cells over 4 biological replicates; Mutant: n=11 cells over 4 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars(*P< 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.
Nesprin-2 accumulates at the front of the nucleus during confined cell migration (2020)
Figure 5

Figure 5. The recruitment of nesprins is due to nesprins moving to the front of the nucleus.

A, Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces. Cross-sections along the direction of migration (yellow) and perpendicular to the direction of migration (blue, 1-4) are shown below.

B, A confocal slice on the surface of the nucleus shown in A indicates that GFP-nesprin-2 co-localizes with actin cables at the surface of the nucleus. Dashed white lines represent the outlines of the constriction.

C, Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope. Dashed white lines represent the outlines of the constriction. The distance between mCh-LAC and GFP-nesprin-2 was measured at the back, at the constriction, at the front near the constriction, and at the center of the front. (N = 17 cells, over 3 biological replicates. The alignment of the two channels was corrected using at least 28 fluorescent beads.)

D, Two-photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus, but not ablation at the back. The nuclei of DN-KASH, which show a decrease of endogenous nesprin at the nuclear envelope (see Figure EV4A), show a dampened rearward movement upon ablation at the front of nuclei. Bars represent the average value. Error bars represent the standard error on the mean. Back: N= 9, Front: N= 30, Cell Front: N = 16, and DNKASH: N=31, 3 biological replicates.

EXPANDED VIEW FIGURES
Lipid droplet dependent fatty acid metabolism controls the phenotype of tumor-associated macrophages (2019)
Figure 1

Figure 1. Oleate polarizes bone marrow-derived myeloid cells into immune suppressive tumor-associated macrophages.

(A) Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with 0.2 mM of the indicated compounds for 7 days. Gr1-CD11b+ cells were sorted and lysed for microarray. The hierarchical clustering was based on the different expression genes between BSA (Con) and oleate group. (B) Signature genes involved in lipid metabolism, dendritic cell maturation, macrophage maturation, tumor associated macrophages (TAMs) phenotype, MHCII complex and innate immune response are listed and (C) validated via flow cytometry or catalytic activity assay. Data are expressed as mean±SD from two to four independent experiments. Unpaired Student's two-tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups. *p<0.05; **p≤0.01
Lipid droplet dependent fatty acid metabolism controls the phenotype of tumor-associated macrophages (2019)
Figure 2

Figure 2. Oleate-induced mitochondrial respiration regulates the suppressive phenotype of myeloid cells.

(A and B) Gr1-CD11b+ cells, polarized by the indicated treatment, were purified for mitochondrial respiration detection. The oxygen consumption rate (OCR) of these cells was monitored after the addition of oligomycin (OA) (1 µM), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA) (0.5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified. (C and D) Forty µM etomoxir were added starting from day 0 of bone marrow polarization, followed by mitochondrial respiration assay by using the same amount of polarized Gr1-CD11b+ myeloid cells on day 7. The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer. (E and F) Exemplary plots of the proportion of CD206+ cells, the mean fluorescence intensity (MFI) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry. (G, H) T cell proliferation assay was performed via co-culture with purified CD4+ T cells in the ratios of M (myeloid cells): T (T cells) = 1:30. (I) NO production from the co-culture supernatant was quantified by Griess reaction. Data are expressed as mean±SD from two to four independent experiments and analyzed by either One-way analysis of variance (ANOVA) (B) or two-way ANOVA with Tukey's post hoc test (D, F, H, I). *p<0.05; **p≤0.01; ***p≤0.001.
Lipid droplet dependent fatty acid metabolism controls the phenotype of tumor-associated macrophages (2019)
Figure 3

Figure 3. Lipid droplet-derived fatty acids facilitate mitochondrial respiration in myeloid cells.

(A) The formation and utilization of lipid droplets in eukaryotes. Five µM combination of DGAT inhibitors (DGAT1 inhibitor A922500 and DGAT2 inhibitor PF-06424439), 40 µM ATGL inhibitor atglistatin (Atg) or 5 µM MAGL inhibitor MJN110 were added to the bone marrow polarization system in the presence of 40 ng/ml GM-CSF and indicated compounds for 7 days. (B) The oxygen consumption rate (OCR) of 1×105 purified Gr1-CD11b+ cells was monitored as described in Figure 2. The basal OCR, basal ECAR (extracellular acidification rate), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified. (C) The polarization state was evaluated via the expression of CD206. (D) T cell proliferation assay was performed employing co-culture with purified CD4+ T cell in the variant ratios. (E) The lipid droplets accumulation was determined by BODIPY staining. The percentage of divided cells and proliferation index were calculated. (F) Nitric oxide (NO) concentration in the co-culture supernatant was quantified by Griess reaction. Shown is the mean±SD from two to four independent experiments and analyzed by two-way ANOVA with Tukey's post hoc test. *p<0.05; **p≤0.01; ***p≤0.001
Lipid droplet dependent fatty acid metabolism controls the phenotype of tumor-associated macrophages (2019)
Figure 4

Figure 4. Polarization of suppressive myeloid cells depends on mTOR signaling pathway.

(A) Bone marrow-derived myeloid cells were treated with BSA-control (Con) or oleate in the presence or absence of 10 nM rapamycin (Rapa). The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression. (B) Purified Gr1-CD11b+ myeloid cells were co-cultured with CD4+ T cells for proliferation assay. The supernatant was collected for nitric oxide (NO) detection. (C) The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified. (D and E) Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells as determined by flow cytometry and (E) lysed for western blot. Shown is the mean±SD from two to four independent experiments. Two-way ANOVA with Tukey's post hoc test was performed to compare the effect of rapamycin in different groups. *p<0.05; **p≤0.01; ***p≤0.001
Lipid droplet dependent fatty acid metabolism controls the phenotype of tumor-associated macrophages (2019)
Figure 5

Figure 5. Disrupting lipid droplet-derived fatty acids in myeloid cells impairs tumor growth.

(A) Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206+ (black) and CD206- (grey) cells. (B) Balb/c mice were inoculated with 5×105 CT26 tumor cells subcutaneously. Since day 6, vehicle or DGAT inhibitors (iDGAT) were administrated via intra-tumoral injection once daily. (C) At day 13, tumor infiltrated immune cells were isolated and stained for CD206 and CD11b. (D) 5×105 MCA205 were injected subcutaneously into C57BL/6 mice. PBS, liposome control or iDGAT-liposome was injected into the peritoneum every other day starting at day 7. Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis. (E) Immune cells from spleen, tumor-draining lymph nodes (TDLN) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells. (F) Tumor infiltrating CD8+ T cells (red) were stained together with nuclei (DAPI/blue) after sacrificing the tumor bearing mice. White bars represents 200 μm. Shown is the mean±SEM from two to four independent experiments. Two-way ANOVA with Tukey's post hoc test was performed to compare the effect of iDGAT-liposome in tumor model. One-way ANOVA was performed to compare the effect of iDGAT-encapsulated liposome in human CD14+ cells. *p<0.05; **p≤0.01
Bim inhibits autophagy by recruiting Beclin 1 to microtubules. (2012)
Figure 1

Bim Physically Interacts with Beclin 1 Independently of the Bim BH3 Domain

(A) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), Beclin 1-Flag, Beclin 1-Flag/HA-Bim(EL), Beclin 1-Flag/HA-Puma, Beclin 1-Flag/HA-Noxa, or Beclin 1-Flag/Bad. Anti-Flag (M2) was used for immunoprecipitation, and proteins were detected with anti-Bcl-xL, anti-HA, or anti-Bad, anti-Flag (Rabbit).

(B) HA-Bim (wild-type) was cotransfected into Bax/Bak double-knockout (DKO) mouse embryonic fibroblasts (MEFs) with empty vector (IP negative control) or Beclin 1-Flag. Immunoprecipitations were performed as in (A).

(C) Beclin 1-Flag was cotransfected into Bax/Bak DKO MEFs with empty vector (IP negative control) or HA-Bim (wild-type). Anti-HA was used for immunoprecipitation. ∗, antibody heavy chain.

(D) HeLa cell lysates were subjected to GFP antibody (negative control) and anti-Bim antibody immunoprecipitation. Proteins were detected with anti-Beclin 1 and anti-Bim. ∗∗, antibody heavy chain; ∗, antibody light chain.

(E) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), wild-type HA-Bim(EL), or HA-Bim L152E F159E [Bim(EL)EE]. Anti-Bim was used for immunoprecipitation. ∗, antibody light chain.

(F) HeLa cells were transfected with vector, wild-type HA-Bim, or HA-BimEE. Lysates were probed with the indicated antibodies. HeLa cells treated with staurosporine (STS) for 5 hr were a positive control. Note that wild-type Bim causes apoptosis, which reduces its levels compared to BimEE.

(G) Vector, wild-type Bim(EL), or Bim(EL) L152E F159E (BimEE) was transfected into HeLa cells. Transfection efficiency was >90%. After 20 hr, cell viability was determined by measurement pf ATP levels. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(H) HeLa cells were transfected with vector/GFP (3:1), wild-type HA-Bim/GFP (3:1), or HA-BimEE/GFP (3:1) (to ensure that Bim-transfected cells also contain GFP). After 16 hr, cells were stained with Annexin V-APC and analyzed by FACS. The percentage of APC and GFP double-positive cells/GFP-positive cells are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(I) Bim(EL)/Bcl-xL were cotransfected into HeLa cells with empty vector (IP negative control) or Beclin 1-Flag; Bim(EL)-EE/Bcl-xL were also cotransfected into HeLa cells with Beclin 1-Flag. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. NS, not significant.

(J) BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag, BimL-EE (L)/Beclin 1-Flag, and BimS-EE (S)/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant.

See also Figure S1.
Bim inhibits autophagy by recruiting Beclin 1 to microtubules. (2012)
Figure 2

Autophagosome Levels in Bim-Depleted and -Overexpressing Cells

(A) Bim−/− and Bim+/+ MEFs were cultured in 6-well dishes. Cells were treated with vehicle or Bafilomycin A1 (Baf) for 4 hr. Blots were probed with the indicated antibodies. LC3-II/tubulin in wild-type control MEFs is set as 1. The relative value of LC3-II/tubulin in Bim-knockout MEFs is shown (n = 5). Data are mean ± SD. ∗∗∗p < 0.001.

(B) HeLa cells were transfected with control siRNA and Bim siRNA. After 48 hr, cells were treated with vehicle or Baf for 4 hr. Blots were probed with the indicated antibodies and analyzed as described in (A). ∗∗∗p < 0.001.

(C) Bim−/− and Bim+/+ MEFs were transfected with GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.

(D) HeLa cells were transfected with control plasmid (pMKO)/GFP-LC3 or pMKO-BimshRNA/GFP-LC3 (to ensure that GFP-LC3-positive cells also contain BimshRNA or control plasmid, the ratio of pMKO or pMKO-BimshRNA/GFP-LC3 is 3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(E) HeLa cells were transfected with control plasmid (pMKO) or pMKO-BimshRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(F) HeLa cells were transfected with control plasmid (pMKO)/GFP-PX or pMKO-BimshRNA/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.

(G) DFCP1-GFP cells were transfected with control siRNA or Bim siRNA. The numbers of GFP puncta were assessed. Data are shown as mean ± SD.∗p < 0.05.

Images represent DFCP1-GFP vesicles in cells transfected with control siRNA and Bim siRNA. Arrows mark DFCP1-GFP puncta.

(H) HeLa cells were treated with control siRNA or Bim siRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.

(I) Bim(EL)/control siRNA, Bim(EL)/Bim siRNA, Bim(EL)EE/control siRNA, Bim(EL)EE/Bim siRNA, vector/control siRNA, and vector/Bim siRNA were transfected into HeLa cells. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.

(J) HeLa cells were transfected with control plasmid or Bim(EL)EE. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.

(K) Bax/Bak DKO MEFs were transfected with control plasmid /GFP-LC3, wild-type Bim(EL)/GFP-LC3 (3:1), or Bim (EL) EE/GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.

(L) Two independent control vector (pcDNA3) or Bim(EL)EE stably expressing HeLa cell clones were transfected with GFP-LC3 respectively. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(M) HeLa cells were transfected with control plasmid/GFP-PX or Bim(EL)EE/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. Fluorescent microscope images show GFP-PX vesicles.

See also Figure S2.
Bim inhibits autophagy by recruiting Beclin 1 to microtubules. (2012)
Figure 3

Autophagy Is Increased in Bim-Knockout Mice

(A) Lysates from spleen-derived T cells of wild-type (Bim+/+) and Bim-knockout (Bim−/−) mice were immunoblotted and probed with indicated antibodies. Statistics were from three knockout and control mice. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(B) T cells from Bim+/+ and Bim−/− mice were cultured with/without Baf for 4 hr preceding harvest, immunoblotted, and probed with the indicated antibodies.

(C) Spleen-derived T cells from Bim+/+ or Bim−/− mice were stained with rabbit anti-LC3 antibody. Representative confocal images and quantification from triplicates (n = 100–110) are shown. Data are shown as mean ± SD. ∗∗∗p < 0.0001.

(D) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗p < 0.01.

(E) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001.

(F) RNA from Bim wild-type or Bim-knockout mouse livers was analyzed by qRT-PCR for p62/beta-actin mRNA. The mean ± SD from three mice per group is shown. NS, not significant.

See also Figure S3.
Bim inhibits autophagy by recruiting Beclin 1 to microtubules. (2012)
Figure 4

The Effect of Bim on Autophagy Is Beclin 1 Dependent

(A) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. Blots were probed as indicated. Data are shown as mean ± SD. ∗∗p < 0.01.

(B) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. After 24 hr, cells were transfected with GFP-PX for 24 hr. GFP-PX vesicles were assessed with a confocal microscope. Data are shown as mean ± SD. ∗∗p < 0.01. Arrows label cells with increased number/size of GFP-PX vesicles.

(C) GFP-mRFP-LC3 HeLa cells were treated with control, Bim siRNA, or Bcl-2 siRNA and then analyzed with Cellomics microscope. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(D) GFP-mRFP-LC3 stable HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA and then analyzed with Cellomics microscope and matching confocal images are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.

See also Figure S4.