EMBO/soda-nel huggingface dataset:
Store-Operated Calcium Entry (SOCE) Controls Innate and Adaptive Immune Cell Function in IBD (2022)
Figure 6

Figure 6. Inhibition of SOCE does not impair the viability, differentiation or function of intestinal epithelial cells.

(A-B) Adult stem cell-derived intestinal epithelial spheroids of wild-type C57BL/6 mice were cultured for 4 days in Wnt-free medium to induce differentiation. Initial viability was determined on day 0. DMSO control (vehicle) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy. Scale bar depicts 100 µm.

(C) Transepithelial electrical resistance (TEER) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2.

(D) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt-free medium to induce differentiation into colonic organoids. Initial viability was determined on day 0. DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2, 4 and 6.

(E) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2.

(F) Relative mRNA expression of markers for enterocytes (ALPI), goblet cells (MUC2), and entero-endocrine cells (CHGA) was quantified by RT-PCR in differentiated human colon monolayers after addition of 1 µM BTP2.

Data information: Data bars in A-D represent the mean ± SD, while E-F represent the mean ± SEM of least 2 independent experiments with 1-4 replicates. Statistical significance was calculated using a paired t-test with a Holm-Sidak correction for multiple comparisons.
Store-Operated Calcium Entry (SOCE) Controls Innate and Adaptive Immune Cell Function in IBD (2022)
Figure 7

Figure 7. Systemic CRAC channel inhibition alleviates colon inflammation in mice.

(A-C) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents (Icrac). (A) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1. (B) Current-voltage (I-V) relationship of ICRAC before and after CM4620 treatment. I-Vs were obtained at the time points indicated by the arrowheads in (A). (C) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration (arrowheads) using the formula 1- (ICM4620 / ICtrl). Data are from 8 cells per conditions and shown as the mean ± SEM.

(D) IBD was induced by i.v. injection of 5 x 105 naïve CD4+ T cells into Rag1-/- host mice followed by oral gavage of mice with 20 mg/KG CM4620 or vehicle (SDD) every other day from days 18-49.

(E) Relative weight loss of mice treated with vehicle or CM4620; the start of treatment is indicated by the white arrow. Data are the mean ± SEM of 9 mice per cohort, statistical analysis by unpaired student's t-test , *p<0.05.

(F) Concentrations of CM4620 in the serum of mice at day 49, data are the mean ± SEM. Statistical analysis by unpaired student's t-test, ***p<0.001.

(G) Representative H&E staining of the distal colon of Rag1-/- mice treated with vehicle or CM4620. Colitis scores of 9 mice per cohort. Each symbol represents one mouse. Statistical analysis by Mann-Whitney U test Data are the mean ± SEM, ***p<0.001.

(H) Number of CD11b+Gr-1+ neutrophils and CD4+ T cells (normalized to grams of tissue) in the colon lamina propria (CLP) of mice on day 49 quantified by flow cytometry. Each dot represents one mouse. Data are the mean ± SEM of 9 mice per cohort.

(I) Frequencies of RORγt+ Th17 cells and Foxp3+ Treg cells in the CLP of mice treated with CM4620 or vehicle. Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.

(J) Frequencies of IFN-γ, TNF-α and IL-2 producing CD4+ T cells isolated from the CLP, restimulated ex vivo with PMA and ionomycin for 4 h (without addition of CM4620 during stimulation in vitro) and analyzed by flow cytometry. Bar graphs represent the percentages of IFN-γ+, TNF-α+, IL-2+ cells in CD4+ cells (top row) and mean fluorescent intensity (MFI) of IFN-γ, TNF-α, IL-2 on CD4+ cells (bottom row). Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.

Data information: Statistical analyses in panels H-J by an unpaired student's t-test: ***p<0.001 **p<0.01 *p<0.05.

EXPANDED VIEW FIGURE LEGENDS
Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy (2020)
Figure 1

Figure 1. Lysosomal degradation of NBR1 persists in an ATG7-independent manner.

(A) Schematic depicting the tf-Reporter system. Both RFP and GFP fluorescence are observed throughout autophagosome formation. Upon autolysosome formation, GFP fluorescence is quenched due to the low pH of the lysosomal lumen. Corresponding changes in red:green ratio are reflective of reporter delivery to autolysosomes.

(B) Dot plot indicating fraction of tf-Reporters delivered to the lysosome by ATG7-independent mechanisms. The median red:green ratio of each population was used to calculate fractional delivery according to the following formula: (ratiosgATG7-ratiosgATG9A)/(ratioNegativeCtl1-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Mean values for each reporter are indicated by a black bar. n=3 for each reporter. p values were determined using a one-way ANOVA (p < 0.0001) with Dunnett's multiple comparison test to compare each sample to the tf-LC3 control. *, p < 0.05; **, p < 0.005; ***, p <0.001; ns, not significant.

(C) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells. See Fig EV1A for validation of genotype.

(D) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally. See Fig EV1C for related images.

(E) Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm. See Fig EV1E for images of additional structures.

(F) Presumptive model for ATG7-independent autophagy. Delivery of NBR1 to the lysosome is dependent on FIP200 (blue) and ATG9A (not shown). In the presence of LC3-lipidation, NBR1 incorporation into autophagosomes is driven by receptor interactions with LC3. In the absence of lipidated LC3, NBR1 is selectively delivered to the lysosome by a largely unknown mechanism.

Data information: See also, Figure EV1.

Source data are available online for this figure.
Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy (2020)
Figure 2

Figure 2. Identification of genes required for ATG7-independent autophagy.

(A) Box plot of average beta scores (similar to log-fold change) for tf-NBR1 flux in indicated deletion cell lines. Median values (central band), inner quartiles (boxed regions), 1.5 interquartile ranges (whiskers), and outliers (dots) are indicated. Representative hits across all three genetic backgrounds are indicated in blue.

(B) Gene correlation plot of average beta scores for lipidation dependent autophagy (tf-NDP52, from (Shoemaker et al, 2019)) and lipidation independent autophagy (average of tf-NBR1 from ATG7KO, ATG10 KO and ATG3 KO cells, Table EV2). Highlighted in red are genes with a beta score > 0.5 across all three lipidation-deficient cell lines. In black are lipidation components. Dashed lines, top 1% of beta scores.

(C) Wild-type K562 cells and indicated deletion isolates expressing Cas9 and tf-NDP52 or tf-NBR1 were transduced with individual sgRNAs for indicated genes. The median red:green ratio of each population was used to calculate the fold-repression according to the following formula: (ratiosgGene-ratiosgATG9A)/(ratioNegativeCtl-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Deeper shades of red indicate stronger suppressor phenotypes. Genes were clustered on the basis of their patterns of genetic interactions.

Data information: See also, Appendix Figure S1.

Source data are available online for this figure.
Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy (2020)
Figure 3

Figure 3. tf-NBR1 flux requires TAX1BP1 in the absence of ATG7.

(A) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). (n > 10,000 cells). See Fig EV2A for validation of genotype.

(B) Representative confocal micrograph (as maximum intensity projections) of ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1. Selected region (white box) of micrograph is shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; merged, white; Hoechst, blue. Scale bars: large panel, 5 µm; small panels, 1 µm. Compare to Fig 1D, Fig EV1C.

(C) Correlative light and electron microscopy (CLEM) of K562 ATG7KO/TAX1BP1KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrow indicates a structure of interest. White box demarcates zoomed area in bottom images. NBR1, green; Hoechst, blue. Scale bar: top panels, 5 µm; bottom panels, 500 nm.

Source data are available online for this figure.
Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy (2020)
Figure 4

Figure 4. TAX1BP1 is required for lysosomal degradation of NBR1 in the absence of LC3 lipidation.

(A) Wild-type and indicated K562 knock-out cells expressing tf-NBR1 were transfected with TagBFP or TagBFP-RavZ, and BFP-positive cells were analyzed for red:green ratio by flow cytometry. The median red:green ratio of each sample was used to calculate flux relative to WT+TagBFP. Bar graphs represent mean +/- SD from three independent experiments. n > 7,500 cells per sample. p values were determined using a repeated-measures one-way ANOVA (p = 0.0001) with Tukey HSD post-test. **, p < 0.005; *, p < 0.05; ns, not significant. See also, EV2D.

(B) K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading. Bar graphs represent mean +/- SD from independent experiments (n=4 {NBR1, NDP52}; n=3 {SQSTM1}). p values for WT vs ATG9AKO was determined using a one sample test (theoretical mean = 1) with Bonferroni correction. All other p values were determined using a one-way ANOVA (p< 0.001) with Tukey HSD post-test. ***, p < 0.001, **, p < 0.01; ns, not significant.

(C) Schematized representation of protease protection assay for detecting vesicle closure. Cells were treated for 18h with 50nM Bafilomycin A1 prior to mechanical lysis. The corresponding cell extracts were treated as indicated prior to being resolved by SDS-PAGE and analyzed by immunoblotting.

(D) Wild-type and indicated K562 knock-out cells were treated as described in (C). The corresponding cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with indicated antibodies. Shown are representative images from one experiment with mean intensity levels from (E) indicated below each image (NBR1: n = 4; TAX1BP1: n = 3, NDP52: n = 4, tubulin: n = 4).

(E) Quantitation of protease-protection data from experiments in (D). Bar graphs show the mean +/- SD of each sample from independent experiments. Trypsin-treated samples were compared using a one-way ANOVA (NBR1: n = 4, p = 0.0001; TAX1BP1: n = 3, p = 0.0098; NDP52: n = 4, p < 0.0001) with Tukey HSD post-test. **, p < 0.01; *, p < 0.05; ns, not significant

See also, Figure EV2I.

Data information: See also, Figure EV2.

Source data are available online for this figure.
Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy (2020)
Figure 5

Figure 5. A heterotypic receptor complex defines an ATG7-independent substrate.

(A) Extracts derived from wild-type and deletion K562 cells expressing tf-NBR1 were normalized for total protein by BCA. NBR1 was immunoprecipitated using GFP-Trap dynabeads. Input and eluate were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.

(B) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 and ATG9AKO cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. In conditions where puncta were not observed, representative cytoplasmic regions were selected to showcase diffuseness of signal. TAX1BP1, SQSTM1, or NDP52, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.

(C) Shown are representative confocal micrographs (as maximum intensity projections) of K562 cells expressing tf-NDP52 (or tf-TAX1BP1) and transduced with sgRNAs targeting ATG9A (sgATG9A) or a negative control (sgControl). Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. See Fig EV3D and E for confirmation of ATG9A deletion.

Data information: See also, Figure EV3.

Source data are available online for this figure.
Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy (2020)
Figure 6

Figure 6. Domain analysis of TAX1BP1 function distinguishes canonical and non-canonical autophagy functions.

(A) Domain organization of TAX1BP1 (NP_006015.4) and derived variants. Coiled-coil regions were defined by PCOILS using a 28 amino acid window and cutoff of 0.5. SKICH, SKIP carboxyl homology domain; CC, coiled-coil; Z1 and Z2, ubiquitin-binding zinc finger (UBZ) domains; N, NBR-interacting domain.

(B) A summary of TAX1BP1 variants' activity in complementation and flux analyses. For complementation analysis, ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 were electroporated with BFP-TAX1BP1 constructs and BFP-positive cells were analyzed for red:green ratio (for underlying data, see Appendix Figure S2A and B). For flux analysis, tf-TAX1BP1 variants were transfected into TAX1BP1KO K562 cells and assessed for red:green ratio by flow cytometry (for underlying data, see Appendix Figure S2C and D). In all experiments, median red:green ratios were used to compare populations.

(C) HEK293T cells were transfected with either myc-TAX1BP1WT or myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.

(D) HEK293T cells were transfected with myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.

Data information: See also, Figure EV4G and H.

Source data are available online for this figure.
Receptor-mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy (2020)
Figure 7

Figure 7. TAX1BP1 recruits FIP200 to induce local autophagosome formation.

(A) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.

(B) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.

(C) Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm.

(D) Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) (see Materials and Methods for details of quantitation). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. n > 200 NBR1 puncta for each biological replicate.

(E) Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. a.u., arbitrary intensity units.

(F) TAX1BP1 drives local autophagosome formation in the context of NBR1 puncta. This activity is independent of LC3 or the ubiquitin-binding domains of TAX1BP1. Rather, a newly defined NBR-interacting domain (or N domain, not shown) drives the association of TAX1BP1 with NBR1 puncta. Subsequent recruitment and clustering of FIP200 by TAX1BP1 induces local autophagosome formation and enforces cargo specificity, thereby replacing the requirement for lipidated LC3.

Data information: See also, Figure EV5.

Source data are available online for this figure.
Streptolysin O and its co-toxin NAD-glycohydrolase protect group A Streptococcus from Xenophagic killing. (2013)
Figure 1

SLO and NADase promote intracellular survival of GAS in oropharyngeal keratinocytes.

A. Intracellular survival of GAS strains 188, 188SLO- (SLO-), and 188NADase- (NADase-). Intracellular CFU were quantified at 2 h, 4 h, 6 h, 8 h, and 24 h, and intracellular survival was calculated as the percent viable CFU compared to 2 h. B. Intracellular survival of GAS strain 188SLO- that was partially complemented from plasmid piSLO (SLO-(piSLO)), strain 188 and 188SLO- that contained the empty complementation vector, 188(pSIV4) and SLO-(pSIV4), respectively. C. Intracellular survival of GAS strain JRS4, JRS4SLO- (SLO-), and JRS4NADase- (NADase-). Experiments were performed in triplicate and values represent the mean of three independent experiments ± SD. *, P<0.001.
Streptolysin O and its co-toxin NAD-glycohydrolase protect group A Streptococcus from Xenophagic killing. (2013)
Figure 2

Xenophagy in oropharyngeal keratinocytes can be stimulated by SLO or SLS and is not associated with killing of intracellular GAS.

A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain. B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material. C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05. D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.
Streptolysin O and its co-toxin NAD-glycohydrolase protect group A Streptococcus from Xenophagic killing. (2013)
Figure 3

Expression of active NADase plays a central role in the intracellular survival of GAS in oropharyngeal keratinocytes.

A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001. NADase western blot (B) and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).
Streptolysin O and its co-toxin NAD-glycohydrolase protect group A Streptococcus from Xenophagic killing. (2013)
Figure 4

SLO, SLS, and NADase affect xenophagy and GAS intracellular survival.

A. Confocal microscopy demonstrating the association between ubiquitin (green) and intracellular GAS (red) at 30 min post-infection in keratinocytes infected with GAS strain 188, 188SLO-, 188NADase-, or 188SLO(Y255A). Extracellular GAS were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments. B. Hemolytic activity of culture supernatants of GAS strain 188, 188SLO-, and 188SLO(Y255A). The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane, demonstrated by the failure of culture supernatants from 188SLO(Y255A) to lyse erythrocytes despite producing wild-type amounts of the variant SLO protein. Inset, corresponding Western blot for SLO in culture supernatants from these strains. C. NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771, 771SLO-, 771SLO(Y255A), or 771NADase-. Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO(Y255A), but reduced in 771SLO-. D. The intracellular survival of 188SLO(Y255A) was significantly lower than that of its parental strain, 188, and similar to that of 188SLO-. *, P<0.01. E. Confocal microscopy demonstrating the association between galectin 8 (green) and intracellular GAS (red) in keratinocytes infected for 30 min with GAS strain 188, 188SLO-, or 188 SLO-SLS-. Extracellular GAS (GAS(out)) were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments. 188SLO-SLS- was never associated with galectin 8 (0%).