EMBO/soda-nel huggingface dataset:
The Ras GTPase-activating-like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL-1beta release via exosomes (2022)
Figure 6

Figure 6. IQGAP1 drives GSDMD-dependent exosomal IL-1β release in vivo.

A. Histology staining for IQGAP1 and GSDMD in naive and inflamed (3 days of DSS-challenge) colon tissues. Scale bar, 100μm.

B. Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls. n=5-7. Data were presented as mean ± SEM. *, P<0.05 by two-tailed unpaired t test.

C. Western blot analysis for supernatant collected from colon explants of DSS-treated wild-type (WT), Gsdmd-/-(GSDMD KO) and Iqgap1-/- (IQGAP1 KO) mice. Supernatant was first pulled down by IL-1β antibody and samples were subjected to Western blot analysis. The indicated proteins were probed.

D. Immunofluorescent staining with anti-GTP-CDC42 in DSS treated colons from Gsdmd+/+ (WT) and Gsdmd-/-(GSDMD KO), IQGAP1+/+ and IQGAP1-/- mice. Data were presented as mean ± SEM. **, P<0.01; ***, P<0.001 by unpaired two-tailed t test.

Data information: All experiments were repeated twice with consistent results. Representative results are shown.
An incoherent feedforward loop interprets NFκB/RelA dynamics for TNF-induced necroptosis decisions (2020)
Figure 1

Figure 1

Necroptosis kinetics are reflective of an incoherent feedforward loop

(A, B) Conceptual mathematical modeling schematics depict TNF-induced necroptosis signaling via RIPK1/3 (RIP) and phosphorylation of MLKL (pMLKL). RIP is counteracted by a putative (A) constitutive, or (B) stimulus-induced, NFκB-dependent survival factor X.

(C, D) Time course simulations of pMLKL levels in 300 single cells where each trajectory crossing a threshold represents a cell death event.

(E, F) Distributions of death times that result from simulations in (C, D), respectively. Fractional survival indicated after 24-hour time course simulation.

(G) Live-cell microscopy workflow and automated image analysis via NECtrack to quantify TNF-induced necroptosis kinetics in L929 cells. Distributions of death times and death rates are computed from raw counts of live and dead cells based on nuclear propidium iodide (PI) staining.

(H) Distribution of death times in TNF-treated L929 wildtype (wt) cells (representative data of three independent experiments).

(I) Normalized death rates in L929 wt cells plotted with pMLKL protein levels measured via immunoblot (mean of three independent experiments ± standard deviation; corresponding images of representative Western blot experiment in Figure EV1F).

(J) Average death rates of the early (<12 hours) and late phase of the TNF time course data in (I) (mean of three independent experiments ± standard deviation; two-tailed Student's t-test ***P<0.001).

(K) Distribution of death times in L929 RelA-knockout (RelA KO) cells treated with TNF (representative data of three independent experiments).

(L) Probability of unimodal distributions of death times calculated by Hartigan's dip significance (mean of three independent experiments ± standard deviation; two-sample t-test **P<0.01).

(M) Death rates in TNF-treated cell lines including clonal RelA-knockout population (mean of three independent experiments ± standard deviation).
An incoherent feedforward loop interprets NFκB/RelA dynamics for TNF-induced necroptosis decisions (2020)
Figure 2

Figure 2

Rapid induction of A20 transiently inhibits the RIPK1-RIPK3 complex and necroptosis

(A) TNF-induced mRNA expression in indicated L929 cell lines measured via qRT-PCR (mean of three independent experiments ± standard deviation, two-tailed Student's t-test *P<0.05, **P<0.01).

(B) Immunoblot after co-immunoprecipitation (IP) of RIPK3. FT, flow through.

(C) Relative quantification of A20 in RIPK3-IP fraction (means and statistical significance established for 0, 2, 4.5 and 8 hour time points from three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, **P<0.01, n.s., P>0.05).

(D) Histogram of volume normalized mRNA copy numbers measured by smFISH of NFκB target genes A20 and IκBα in L929 wildtype (wt) cells treated with TNF (representative data of three independent experiments; two additional independent experiments in Figure EV4H).

(E) Fractions of "responder" cells (A20 count > 1 per cell) (mean of all three independent experiments ± standard deviation).

(F) Fractional survival of wt cells after the early (<12 hours) and late phase of TNF time course obtained by microscopy (mean of three independent experiments ± standard deviation).

(G) A20 mRNA expression via qRT-PCR in TNF-treated wt cells transfected with targeting (siA20) or non-targeting (siCon) siRNA (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05).

(H) Immunoblot and relative quantification of basal A20 protein after siRNA-knockdown normalized to non-targeting siRNA control (mean of three independent experiments ± standard deviation; two-tailed Student's t-test revealed no statistically significant difference between targeting and non-targeting siRNA treatment, n.s., P>0.05).

(I,J) (I) TNF-induced distributions of death times (representative data of three independent experiments), and (J) death rates obtained by live-cell microscopy (mean of three independent experiments ± standard deviation).
An incoherent feedforward loop interprets NFκB/RelA dynamics for TNF-induced necroptosis decisions (2020)
Figure 3

Figure 3

The NFκB-A20-RIPK3 incoherent feedforward loop discriminates TNF stimulus dynamics

(A) Modeling schematics depict TNF-induced activation of complex I (C1) and IKK to induce transcriptional activity of NFκB. C1 can also initiate activation of RIPK1 and RIPK3 to induce phosphorylation of necroptosis executor MLKL (pMLKL). TNF-induced expression of IκB attenuates NFκB, whereas A20 inhibits IKK and RIPK3.

(B,C) Computational simulations and microscopy analysis of death time distributions (left, representative data of three independent experiments) and death rates (right, mean of three independent experiments ± standard deviation) in TNF-treated L929 (B) wildtype (wt), or (C) RelA-knockout (KO) cells.

(D) Simulated death time distributions with 2- or 4-fold increased constitutive A20 expression in the absence of inducible transcription (300 simulated cells per condition).

(E) Death time distributions in TNF-treated parental RelA KO cells (-) or RelA KO cells expressing A20 from a constitutive transgene (pBabe-A20, +, representative data of three independent experiments).

(F) Average death rates (<12 hours) in TNF-treated cells (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, or no statistically significant difference, n.s., P>0.05).

(G,H) (G) Distribution of death times (representative data of three independent experiments), or (H) death rates in TNF-treated wt, parental A20 KO cells, or A20 KO cells reconstituted with an NFκB-inducible transgene (fIL8-A20; mean of three independent experiments ± standard deviation).

(I,J) (I) Simulations and (J) experimental measurements of 24-hour fractional survival after varying durations of transient or sustained (24h) TNF stimulation (mean of three independent experiments ± standard deviation; two-tailed Student's t-test ***P<0.001, or no statistically significant difference, n.s., P>0.05).
An incoherent feedforward loop interprets NFκB/RelA dynamics for TNF-induced necroptosis decisions (2020)
Figure 4

Figure 4

Dysregulated NFκB dynamics diminish the cellular discrimination of TNF exposures

(A) Simulations of A20 mRNA concentrations in versions of the NFκB-necroptosis model where expression is under the control of synthetic NFκB activity following step functions of 0.5, 1, 2, 4, 8, or 16 hours duration (smoothed line is population average, and shaded area the 30th percentile around the median).

(B) Fractional survival that results from simulations in (A).

(C) Immunoblot for IκBα and IκBε in L929 wildtype and CRISP/Cas9-knockout cell lines. Asterisks depict unspecific bands.

(D) Normalized RelA activity dynamics after TNF treatment quantified via EMSA (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05, **P<0.01; corresponding images of representative experiment in Figure EV4A).

(E) A20 mRNA quantified via qPCR (mean of three independent experiments ± standard deviation; two-tailed Student's t-test *P<0.05).

(F) Immunoblot for A20 (representative data of three independent experiments).

(G) Fractional survival after 24 hours of TNF treatment in indicated cell lines (mean of three independent experiments ± standard deviation; two-tailed Student's t-test **P<0.01).

(H) Death rates in TNF-treated indicated cell lines including isogenic IκBα/IκBε-knockout population (mean of three independent experiments ± standard deviation).

(I) Death rates in TNF-treated cell lines treated with targeting (siA20) or non-targeting (siCon) siRNA (mean of three independent experiments ± standard deviation).

EXPANDED VIEW FIGURE LEGENDS
MECHANICAL COORDINATION IS SUFFICIENT TO PROMOTE TISSUE REPLACEMENT DURING METAMORPHOSIS IN DROSOPHILA (2019)
Figure 1

Figure 1. Ventral nests growth

A) Ventral nests expansion at 0 min (onset); 245 min, when ventral and spiracle nests fuse; 515 min, when intersegment nests fuse; and 750 min, when most LECs are gone. We employed for these analyses the following stock: w; If/CyO; ZCL2207::hh-DsRed/MKRS. The green signal corresponds to ZCL2207 (Septate junction GFP marker) outlining all apical cell membranes and the red signal to nuclei from the posterior compartment expressing RFP under the control of the hedgehog promoter. Arrows point to the spiracular and ventral nests on the top panel and to the junction between these two nests in the second panel. Images of confocal sections have been rotated to maintain consistent alignment of the dorso-ventral axis (dorsal is up). The outlines of the confocal images are marked with a yellow dotted line. Scale bar = 40 µm.

B) Comparison of manual vs. PIV surface area kinetics [evolution of nests (red - manual, black - PIV) and LECs (cyan - manual, blue -PIV)] (averaged from 6 movies - see Appendix Figure S1).

C) Nests rate of growth from a representative single nest. Note the similarity in behavior (red - manual, black - PIV) in three sequential phases: Phase 1 (Ph 1), accelerated growth; Phase 2 (Ph 2), sustained growth; and Phase 3 (Ph 3) decelerated growth).

D) Progression of the elongation ratio (red - manual, black - PIV) of nests (averaged from 6 movies). Note the initial fast decay on the elongated shape of the nests.

E) Nest orientation evolution (averaged from 6 movies) (red - manual, black - PIV). Anterior is π (3.1415 rads, dorso ventral is π/2 (1.57 rads) and posterior is 0.
MECHANICAL COORDINATION IS SUFFICIENT TO PROMOTE TISSUE REPLACEMENT DURING METAMORPHOSIS IN DROSOPHILA (2019)
Figure 7

Figure 7. Density power and surface stress upon interference in histoblasts growth or LECs delamination

A) Density power radial kymograph of a representative time-lapse movie of the nests expansion process after blocking histoblasts proliferation by overexpressing Dacapo. Each horizontal line displays at each time point (left scale) the mean of the power value at every distance (bottom scale) to the nest-LECs border (vertical black line). Each phase is represented in a colored background with three different densities of purple. Power values color scale is on the right.

B) Comparison of the means and standard deviations of the density power radial kymographs in phase 3 for Dacapo overexpressing nests (green) and wild type (purple).

C) Surface Stress radial kymograph of a representative time-lapse movie as in A. Phases are represented in different densities of brown. Stress values color scale is on the right.

D) Comparison of the means and standard deviations of the surface stress radial kymographs in phase 3 for Dacapo overexpressing nests (green) and wild type (brown).

E) Density power radial kymograph of a representative time-lapse movie after blocking LECs delamination by overexpressing P35. Power values color scale is on the right.

F) Comparison of the means and standard deviations of the density power radial kymographs in phase 3 for P35 overexpressing LECs (green) and wild type (purple).

G) Surface Stress radial kymograph of a representative time-lapse movie as in E. Phases are represented in different densities of brown. Power values color scale is on the right.

H) Comparison of the means and standard deviations of the surface stress radial kymograph in phase 3 for P35 overexpressing LECs (green) and wild type (brown).
MECHANICAL COORDINATION IS SUFFICIENT TO PROMOTE TISSUE REPLACEMENT DURING METAMORPHOSIS IN DROSOPHILA (2019)
Figure 8

Figure 8. Surface stress patterns

A) Laser microsurgery was performed at the different sides of the ventral nests during phase 2 (300 minutes) in parallel cuts [in the LEC tissue at 40 µm of the nests/LEC border (cyan) and within the nests at 10 µm (red) and 40 µm (green) of the edge]. Myo-GFP transgenic pupae were used to visualize the cortex displacements. This example illustrates at the anterior edge the laser cuts positions. Scale bar = 15 µm.

B) Cortex recoil velocity (pixels per second) at different time points after laser cutting (+ 10 sec) for a single experiment. Note the exponentially decaying speed.

C) Inferred stress differences (brown) and mean recoil velocities (blue) as measured by PIV and in the LECs (cyan - n = 18)), 10 µm (red - n = 16) and 40 µm within the nest (green - n = 20). Error bars represent Standard Deviations.

D) Snapshots of a laser cut in the nest area. PIV arrows are shown in red. Scale bar = 5 µm.

E) Comparison of the mean laser recoil velocities in phase 3 for Dacapo overexpressing nests (green) with wild type (blue) at different distances (-40 - n = 16, -10 - n =22, +40 - n = 14) as in C. Error bars represent Standard Deviations.

F) Comparison of the mean laser recoil velocities in phase 3 for P35 overexpressing nests (green) with wild type (blue) at different distances (-40 - n = 17, -10 - n = 20, +40 - n = 15) as in C. Error bars represent Standard Deviations.

EXPANDED VIEW FIGURE LEGENDS
Nanog maintains stemness of Lkb1-deficient lung adenocarcinoma and prevents gastric differentiation (2020)
Figure 1

Figure 1 Identification of Nanog as the stemness factor in KL lung tumors.

(A, B) Gene Set Enrichment Analysis (GSEA) identifies Stem-cell like expression signature in KL tumors compared with KrasG12D/+ (K) lung tumor (A) or KrasG12D/+; P53-/- (KP) lung tumors (B). Significance was calculated by permutation test.

(C) Heat map demonstration of genes that were mostly enriched in the pathway of pluripotency of stem cells. The color bar showed the relative RNA-seq signal (Z-score of normalized FPKM).

(D) The protein abundance of NANOG was upregulated in KL tumors compared with K and KP mice. Scale bar, 50 μm.

(E) Scheme illustrator of mouse model.

(F-H) Histological examinations of tumor types in (F) K versus KrasG12D; NanogL/L (KN), (G) KP versus KrasG12D; P53L/L; NanogL/L (KPN) and (H) KL versus KrasG12D; Lkb1L/L; NanogL/L (KLN). Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom).
Nanog maintains stemness of Lkb1-deficient lung adenocarcinoma and prevents gastric differentiation (2020)
Figure 2

Figure 2 Nanog deletion triggers gastric differentiation in KL model.

(A, B) Human (A) and KLN murine (B) lung sections were stained with H&E or with Periodic Acid Schiff (PAS) and Alcian blue (AB). Scale bar, 50 μm.

(C) Statistical analysis of mucinous tumor numbers (no.) in KL (n = 7) or KLN (n = 7) mice. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. Results were shown as mean. **p=0.0066.

(D) Representative pictures of lung tumors at 4 weeks and 8 weeks post Ad-Cre infection in KLN mice. Scale bar, 50 μm.

(E) Statistical analysis of IMA ratio at 4 weeks and 8 weeks post Ad-Cre treatment (n=5 per group). Results were shown as mean ± SEM.

(F) Statistical analysis of ratio of mucinous phenotype in AAH, AD, and ADC (n=5 per group). Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. *p=0.0215.

(G) qPCR analysis of stomach-restricted gene set between KL ADC and KLN mucinous tumor sections (n=3 or 4 per group). Results were shown in a box and whisker plot with the minimum value, first quartile, median, third quartile and maximum value. The box represented the 50% of the central data, with a line inside represented the median. The significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p=0.0260 (Hnf4α), *p=0.0275 (Vsig1), *p=0.0122 (Gkn3), *p=0.0324 (Ctse), **p=0.0084 (Onecut2).

(H) Representative histological and IHC staining for HNF4α, GKN1, NKX2-1, MUC5AC, CK20 in lung tumors derived from ADC and IMA. Scale bar, 50 μm.
Nanog maintains stemness of Lkb1-deficient lung adenocarcinoma and prevents gastric differentiation (2020)
Figure 3

Figure 3 Frequently concurrent loss of NANOG, LKB1 and NKX2-1 expression in human IMA.

(A) Representative images of low, medium, high protein expression of NANOG in human lung adenocarcinomas. Scale bar, 150 μm (top), 50 μm (bottom).

(B) Representative H&E and IHC staining for NANOG in human ADC and IMA. Scale bar, 50 μm.

(C) Statistic analysis of NANOG expression in ADC and IMA. The tumor tissues were analyzed by IHC staining and graded as low, medium, high expression. Significance was calculated by χ2 test for trend.

(D) Western blot for NANOG and TUBULIN in 7 paired human normal lungs (N) and ADC samples (T) and human ESCs. Mr, relative molecular mass. kDa, killoDalton.

(E) Western blot for NANOG and TUBULIN in human adenocarcinoma cell lines transfected with empty vector or human NANOG overexpression vector. The arrow indicated the band of exogenous human NANOG. The asterisk indicated the band of endogenous human NANOG. Mr, relative molecular mass. kDa, killoDalton.

(F) Representative images of low, medium, high protein expression of LKB1 in human lung adenocarcinomas. Scale bar, 150 μm (top), 50 μm (bottom).

(G) Representative H&E and IHC staining for LKB1 in human ADC and IMA. Scale bar, 50 μm.

(H) Statistic analysis of LKB1 expression in ADC and IMA. The tumor tissues were analyzed by IHC staining and graded as low, medium, high expression. Significance was calculated by χ2 test for trend.

(I) The expression grade of IHC staining of NANOG, NKX2-1, LKB1 in 76 human IMA (1 sample/row).
Nanog maintains stemness of Lkb1-deficient lung adenocarcinoma and prevents gastric differentiation (2020)
Figure 4

Figure 4 Blockage of Notch signaling prevents mucinous differentiation in KLN mice.

(A) Relative transcripts of components of Notch signaling, Wnt signaling, Hedgehog signaling pathway in IMA tumors of KLN group relative to KL mice (n=4 per group). Results are mean ± SD. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. **p=0.0048 (Hes1), ***p=0.0003 (Hes5), **p=0.0051 (Hey1), **p=0.0025 (Hey2), *p=0.0110 (Ccnd1).

(B-D) H&E staining of tumors, IHC staining (B) and quantification of NICD (C) and HES1 (D) in KL ADC and KLN IMA. Representative images were shown. Scale bar, 50 μm. Results were shown as mean ± SD. The numbers of tumors analyzed over KL ADC and KLN IMA were 15, 18 for NICD, 16, 15 for HES1. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p=0.0188 (C), **p=0.0040 (D).

(E) Representative H&E of tissues and IHC staining for NICD in human ADC and IMA. Scale bar, 50 μm.

(F) Statistic analysis of NICD expression in human IMA relative to ADC. The tumor tissues were analyzed by IHC staining and graded according as low, medium or high expression. Significance was calculated by χ2 test for trend.

(G, H) Statistical analysis of NICD (G) and HES1 (H) expression in lung sections of KLN mice following vehicle or LY-411575 treatment. The numbers of tumors analyzed over vehicle and LY-411575 groups were 22, 19 for NICD, 22, 22 for HES1. Results were shown as mean ± SD. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. ****p<0.0001.

(I) H&E and IHC staining of NICD and HES1 in lung tumor tissues of vehicle and LY-411575 groups. Scale bar, 50 μm.

(J) H&E, AB and PAS staining in lung tumors of vehicle and LY-411575 groups. Scale bar, 50 μm.
Nanog maintains stemness of Lkb1-deficient lung adenocarcinoma and prevents gastric differentiation (2020)
Figure 5

Figure 5 Mucinous tumors from KLN model are resistant to phenformin treatment.

(A) Statistic analysis of ADC numbers of vehicle (Veh) or phenformin (Phen) treatment group in KL mice (n=4 in Veh group, n=3 in Phen group). Results were shown as mean ± SD.

(B) IHC staining of Ki67- and CC3-positive cells in lung ADC of vehicle and phenformin (Phen) groups. Scale bar, 50 μm.

(C, D) Statistical analysis of Ki67 (C) and CC3 (D) activity in ADC lung sections of vehicle (Veh) and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle and phenfortmin groups were 25, 22 for Ki67, 32, 23 for CC3. Results were shown as mean ± SEM. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. ****p<0.0001 (C), *p=0.0162 (D).

(E) Statistic analysis of ADC numbers of vehicle (Veh) or phenformin (Phen) treatment group in KLN mice (n=7 per group). Results were shown as mean ± SD. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. *p<0.05.

(F) Representative photos of H&E, AB and PAS staining in IMA treated with phenformin (Phen). Scale bar, 150 μm. Inset: Representative enlarged photos of lung lobe of KLN mice treated with phenformin. Scale bar, 50 μm.

(G) Statistic analysis of IMA numbers of vehicle or phenformin (Phen) treatment group in KLN mice (n=11 per group). Results were shown as mean.

(H) IHC staining of Ki67- and CC3-positive cells in murine lung ADC and IMA of vehicle and phenformin (Phen) groups. Scale bar, 50 μm.

(I, J) Statistical analysis of Ki67 (I) and CC3 (J) activity in ADC and IMA lung sections of vehicle and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17, 8, 9 for Ki67, 10, 10, 7 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0010 (I), ***p=0.0009 (I), *p=0.0427(J).