EMBO/SourceData huggingface dataset:
Figure 4Cytochalasin D-treated BMDMs were stained with the membrane-permeable dye Oregon Green 488 Bapta-1 AM to visualize intracellular Ca2+, infected with aggregates of Mtb Erdman WT and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Oregon Green 488 Bapta-1 AM fluorescence values at each time point were normalized to time 0 for uninfected bystander cells and to the time of first contact with an Mtb aggregate for infected cells. (A) Examples of BMDMs dying with (yellow outline) or without (white outline) interacting with extracellular Mtb aggregates. Cell outlines are based on brightfield images Cell death is indicated by Draq7 nuclear staining. Scale bar, 10 μm. (B) Oregon Green 488 Bapta-1 AM fluorescence over time in dying bystander macrophages (n=15) and in dying macrophages in contact with an Mtb aggregates (n=63). Lines represent median fluorescence values for all cells, error bars represent interquartile ranges. The distributions of the fluorescence values at time of death in bystander and dying macrophages are significantly different, p value <0.0001, calculated using a Welch's t test.(E) BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. Percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate without (untr.) or with supplementation of BAPTA-AM. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 60 cells per replicate). Bars represent means and standard deviations, P value calculated using a t test.
Figure 5BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided in Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel (A) Percentage of dead uninfected bystander macrophages (byst.) or dead macrophages in contact (cont.) with an extracellular Mtb aggregate that stain positive for cleaved Caspase-8 (cCasp8). Each symbol represents the percentage of positive dead macrophages for a single biological replicate (n ≥ 50 cells per replicate). Bars represent means and standard deviations. P values were calculated using a t test. (B) Percentage of dead uninfected bystander macrophages (byst.) or dead macrophages in contact with an extracellular Mtb aggregate (cont.) that display ASC-specks. Each symbol represents the percentage of positive dead macrophages for a single biological replicate (n ≥ 80 cells per replicate). Bars represent means and standard deviations. P values were calculated using a t test.BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel (C) Representative example of a dead macrophage in contact with an extracellular Mtb aggregate that display a ASC-speck (white arrow). Nuclei stained with Hoechst. Scale bar, 10 μm.BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel Median fluorescence values for bystander macrophages (byst.) or macrophages in contact with an extracellular Mtb aggregate (cont.) stained with anti-cleaved Caspase-1 antibody (cCasp1), an (E) anti-phosphorylated RIP3 antibody (pRIP3) or (F) anti-phosphorylated MLKL antibody (pMLKL). In (G) BMDMs were treated with MCC950 (NLRP3 inhibitor) or with VX765 (Caspase-1 inhibitor) during the course of the infection. Each symbol represents a single macrophage. Black bars represent median and interquartile range. P-value were calculated using an unpaired Mann-Whitney test; ns, P values > 0.05. E: n=182, 112 respectively; F: n=186, 84 respectively; G: n=117, 121, 111, 129 respectively; n represents the number of individual cells analyzed per condition).
Figure 6(A-D) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. The plots represent the percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P-values were calculated using a one-way ANOVA test (A,C,D) or t test (B) comparing each strain to the wild-type reference strain (A-D); ns, P values > 0.05. (A) Macrophages in contact with aggregates of Mtb H37Rv wild-type (WT), ∆RD1 mutant and complemented ∆RD1 mutant (∆RD1::2F9). (B) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or PDIM-deficient (fadD26) strains. (C) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or mutant strains (espI eccD1, esxA, espA) and the complemented strains (esxA::esxBA, espA::espA). (D) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or mutant strains (espB, espA espB) and the espB-complemented strains (espB::espB, espA espB::espB).(F) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains, incubated with Annexin V and imaged by time-lapse microscopy at 1-hour intervals for 48 hours. Percentage of macrophages that show Annexin V - positive membrane domains within the first 12 hours after entering in contact with Mtb aggregates. Each symbol represents a single biological replicate (n ≥ 3 replicates with ≥ 90 cells per replicate). Bars represent means and standard deviations.(G) Cytochalasin D-treated BMDMs were infected with aggregates of Mtb Erdman WT, stained with the membrane-permeable dye Oregon Green 488 Bapta-1 AM to visualize cytosolic Ca2+ and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Oregon Green 488 Bapta-1 AM fluorescence values at each time point were normalized to the time of first contact with an Mtb aggregate for infected cells. Values in the plot correspond to the time of death after first contact or 16 hours post-contact for cells that survive. Each symbol represents a single macrophage. (n=123, 64, 48, 71, 78, 55 respectively). Black bars represent median and interquartile range. P-value were calculated using a one sample Wilcoxon test; ns, P values > 0.05.(H) BMDMs were treated as in (A-D). Percentage of macrophages that die within the first 12 hours after interaction with an Mtb aggregate. Infected cells are incubated with different concentration of BTP15 (0, 2, 10, 50 μM) during the course of the experiment. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P-values were calculated using a one-way ANOVA test comparing the treated vs. untreated samples.
Figure 4(A, B) Representative (A) photographs of fly eyes and (B) quantitation with siRNA mediated knockdown of SRPK1 (dSRPK1); n=30-34/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.(C) Representative photographs of fly eyes expressing GMR-GAL4 driven (GGGGCC)28-EGFP with siRNA mediated knockdown of SRPK1 or disruption by insertion. (D) Quantitation of rough eye phenotypes. t-test with Welch corrections for comparisons with the control; n= 31-34 flies/ genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.(E) Representative external eye imaging for detection of GFP aggregates caused by (CGG)90-EGFP transgene expression (top). Converted images used to quantify total intensity of GFP puncta (bottom). (F) Depletion of SRSF1 or SRPK1 by RNAi results in reduced (CGG)90-EGFP puncta compared to control siRNA as quantified by total intensity (a.u. = arbitrary unit). n=13-15 flies/genotype. Error bars represent mean +/- SD. Data information: Statistical analysis was performed using two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001; ∗∗∗∗
Figure 1(A) TNF concentrations in supernatants of WT and Tnf-/- BMDMs unstimulated or stimulated with 10 ng/ml Pam3CSK4, quantified by ELISA. (Left) 0 h, 4 h and 8 h time points. (Right) Close-up of 0 h time point. Dashed line indicates mean of blank measurements. Data are represented as mean ± SD of 3 (4 h, 8 h) or 5 (0 h) biological replicates using cells from 2 (4 h, 8 h) or 3 (0 h) mice. Statistical significance was determined by two-tailed paired-sample t-test.(E - J) Nuclear localization of mVenus-RelA upon stimulation with 10 ng/ml TNF (E - G) or 10 ng/ml Pam3CSK4 (H - J), measured over time in WT and Tnf-/- BMDMs by fluorescence microscopy and quantified by automated image analysis. (E, H) Each row of the heatmap represents the NFκB signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from a single representative experiment is depicted. (F, I) Indicated dynamic features were calculated from signaling trajectories. Violin plots depict pooled data from 8 (F) or 6 (I) biological replicates (total # of cells: 5769/5238 (F), 3843/3899 (I)). Marker indicates median of distribution. Statistical significance was determined by Wilcoxon rank sum test. Number indicates log10(p-value). (G, J) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment. Data are represented as mean ± SD of 8 (G) or 6 (J) biological replicates. Statistical significance was determined by two-tailed paired-sample t-test. (F, G) Some replicates for Tnf-/- BMDMs overlap with data
Figure 2(B) IκBα mRNA expression in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by RT-qPCR. IκBα expression was normalized to GAPDH expression and then to maximum WT value in each time course replicate. Data are represented as mean ± SD of 5 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Some replicates for Tnf-/- BMDMs overlap with dataPhosphorylation of IKK, JNK, ERK, and p38 and total IKK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by SDS-PAGE followed by Western Blotting. (C) One of three representative experiments is shown. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.Phosphorylation of IKK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h For quantification of p-IKK (D) levels, basal levels (0 min) were deducted per genotype before normalizing to maximum WT value in each experiment. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.Phosphorylation of JNK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, p-JNK (E) levels, basal levels (0 min) were deducted per genotype before normalizing to maximum WT value in each experiment. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.
Figure 3(E) TNF receptor 1 (TNFR1, left), TNF receptor 2 (TNFR2, center), and CD11b (right) cell surface expression in Tnf-/- compared to WT BMDMs unstimulated or stimulated for 30 min with 10 ng/ml TNF, measured by flow cytometry. Per experiment, median fluorescence intensity average of 3 culture replicates in Tnf-/- cells is expressed as percent of expression in WT cells. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed one-sample t-test using a hypothesis mean of 100.(F) (Left) TNF receptor 1 (TNFR1) cell surface expression at baseline in BMDMs pre-treated with TNFR1 siRNA, control (ctrl) siRNA, or untreated (-). Median fluorescence intensity is expressed as percent of expression in untreated WT cells. (Right) Percentage of cells with oscillatory NFκB activity (measured by fluorescence microscopy) among all responder cells upon indicated siRNA pre-treatment and stimulation with 10 ng/ml TNF. Per condition, 1032 - 1484 responding cells were analyzed. (Left, right) Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by one-tailed paired-sample t-test.
Figure 4(D) IκBα mRNA expression in Tnf-/- and Tnf-/-p100-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by RT-qPCR. IκBα expression was normalized to GAPDH expression and then to maximum WT value also obtained with each replicate. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Data for Tnf-/- BMDMs is also included(E) Levels of nuclear NFκB DNA binding activity in Tnf-/- and Tnf-/-p100-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by EMSA. Quantification of the NFκB-DNA complex was normalized to the quantified NFY-DNA complex, and then to maximum WT value also obtained with each replicate. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Data for Tnf-/- BMDMs is also shown(F - H) Nuclear localization of mVenus-RelA upon stimulation with 10 ng/ml TNF was measured over time by fluorescence microscopy in Tnf-/- and Tnf-/-p100-/- BMDMs and quantified by automated image analysis. (F) Each row of the heatmap represents the NFκB signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from a single representative experiment are depicted. (G) Indicated dynamic features were calculated from signaling trajectories. Violin plots depict pooled data from 6 biological replicates (total # of cells: 4030/4133). Marker indicates median of distribution. Statistical significance was determined by Wilcoxon rank sum test. Number indicates log10(p-value). (H) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment. Data are represented as mean ± SD of 6 biological replicates. Statistical significance was determined by one-tailed paired-sample t-test. (G, H) Data for Tnf-/- BMDMs is also included
Figure 5(E) Volcano plot of single-cell gene expression in Tnf-/- vs. WT BMDMs upon TNF stimulation. Dashed lines indicate p-value threshold of 0.01 and absolute log2(FC) threshold of 0.5. Only genes induced by P3C4 or TNF stimulation over mock in WT cells are included (p-value threshold of 0.05 and log2(FC) threshold of 0.25).(F) Violin plots of single-cell gene expression in WT and Tnf-/- BMDMs upon mock, TNF, or Pam3CSK4 stimulation. The 10 genes shown are significantly upregulated in Tnf-/- vs. WT cells upon TNF stimulation (see (E)).
Figure 6(D) Violin plot of log­2(FC) of normalized H3K4me1 ChIP-seq signal in TNF- or Pam3CSK4-stimulated over unstimulated WT or Tnf-/- BMDMs within Cluster 1 regions (533 regions). Statistical significance was determined by Kolmogorov-Smirnov test. Data are represented as mean of two biological replicates for each genomic region. In the boxplot, central band indicates the median, boxes the interquartile range (IQR), whiskers values within 1.5 IQR from the median.(E) Examples of genomic regions with differential H3K4me1 signal in TNF stimulation in Cluster 1. Tracks for WT and Tnf-/- BMDMs unstimulated or stimulated with TNF or Pam3CSK4 in two biological replicates (R1, R2) are shown.
Figure 1(B) Coomassie-stained SDS-PAGE of the SEC fractions highlighted in (A) with a horizontal dashed line on top.(C-D) ITC measurements of purified CEP19 titrated with CrRabL2 in the presence of GTP (C) or GDP (D).(G) SEC profile (top) and Coomassie stained SDS gel (bottom) of the complex between RabL2 and a minimal binding-region of CEP19 (residues 107-195) demonstrating a direct interaction.
Figure 2(A) SEC profile of CrRabL2Q83L co-purification with CrIFT81/74/27/25/22 (top) in the presence of non-hydrolysable GTP homologue, GTPγS. Coomassie stained SDS gel of SEC fraction highlighted by dashed lines (bottom). (B) SEC profile for the purification of the HsIFT81/74/27/25/22/RabL2 complex in the presence of GTPγS (top) and the corresponding Coomassie stained SDS gel (bottom).(E-H) GTPases assays with the indicated proteins following the release of inorganic phosphate upon GTP hydrolysis as a function of time. Each experiment was carried out in three technical replicates. Concentrations for experiments in (E) were 55µM CrIFT-B1 pentamer/CrRabL2, 60µM CrIFT-B1 pentamer and 70µM CrIFT-B1 tetramer and 1mM GTP. Concentrations for experiments in (G) were 60µM protein and 30µM GTP. The curves represent averages and error bars indicate standard deviation of measurements for every 100s for panel (E) and every 200s for panel (G). (F, H) Quantification of the reaction rates (arbitrary units of Absorbance (Abs) per second(s)) for each experiment shown in panels (E) calculated using linear regression of the first 500s and (H) using single exponential fit, and agreement with the fit to the curves in (E, G) are indicated by the error bars and R2-value.
Figure 3(A) SEC profile showing that a minimal CrIFT81460-533/CrIFT74460-532 complex co-purifies with CrRabL2Q83L in the presence of GTPγS (left). The right panel displays the Coomassie stained SDS gel of SEC fractions (horizontal top dashed line).(B) An N-terminal hexa-histidine tagged CrIFT81460-533/74460-532 complex interacts with untagged CrRabL2Q83L in a GTPγS-dependent manner in pulldown assays.(E) Quantification of GTPase reaction rates (arbitrary units of Absorbance (Abs) per second(s)) using single exponential fit (D), error bars and R2-value indicate the agreement of the fit to the curves in (D).
Figure 5(C) SEC profile showing that human RabL2B co-purifies with Chlamydomonas IFT-B1 pentamer (left). Coomassie stained SDS gel of SEC fractions highlighted with dashed lines on the SEC profile (right).(E) Quantification of the reaction rates of the GTPase reaction based on single exponential fit of curves in (D) error bars and R2 indicate the agreement of the fit.
Figure 6(B) Affinity pull-down of CrRabL2 with His-tagged WT or mutant CrIFT74T484R/81 complex in the presence or absence of nucleotides.(E) Immunoblot (IB) analysis of expression of IFT74, IFT81, RabL2B and α-tubulin in control (sgSafe) or IFT74 KO RPE cells stably expressing either empty vector, FLAG-IFT74, or FLAG-IFT74 T438R. Representative images from three experimental replicates are shown. The cells were grown to confluence (without serum starvation), lysed and analyzed by Immunoblot. Molecular weights (kDa) estimated from a protein marker are indicated. Red asterisk: endogenous IFT74; black asterisks: FLAG tagged IFT74.(F) Quantification of the immunoblots of IFT74 shown in (E). Data are combined from three independent experiments. Error bars represent ± SD. Statistics obtained through one-way analysis of variance (ANOVA) with Tukey's multiple comparison test.(G-I) Box plots showing centrosomal signal intensity of FLAG (G), IFT81 (H), or RabL2 (I) in the cells described in (D) serum starved for 24 hours. The relative fluorescence signal intensity compared with the average of the control (IFT74 KO + FLAG-IFT74 WT for (G) and sgSafe + empty for (H) and (I)) are shown. Data are combined from three experimental replicates. Statistics obtained through nested one-way ANOVA with Šídák's multiple comparison test. (G) The number of mother centrioles analyzed in each sample per replicate are: 78, 77, 78 (sgSafe + empty); 102, 95, 101 (IFT74 KO + empty); 54, 77, 85 (IFT74 KO + FLAG-IFT74 WT); 49, 83, 99 (IFT74 KO + FLAG-IFT74 T438R), (H) The number of mother centrioles analyzed in each sample per replicate are: 93, 137, 138 (sgSafe + empty); 109, 133, 193 (IFT74 KO + empty); 62, 146, 131 (IFT74 KO + FLAG-IFT74 WT); 41, 143, 133 (IFT74 KO + FLAG-IFT74 T438R), (I) The number of mother centrioles analyzed in each sample per replicate are: 64, 101, 106 (sgSafe + empty); 84, 105, 129 (IFT74 KO + empty); 52, 96, 121 (IFT74 KO + FLAG-IFT74 WT); 73, 92, 102 (IFT74 KO + FLAG-IFT74 T438R).(K) Box plots showing the quantification of number of IFT88 particles per cilium in the experiment described in (J). Data are combined from three experimental replicates: The number of cilia analyzed in each sample per replicate are: 20, 20, 18 (sgSafe + empty); 20, 20, 18 (IFT74 KO + WT); 20, 20, 19 (IFT74 KO + FLAG-IFT74 WT). The number of IFT particles in individual cells in each experiment are available from Figures S7A, D, and G. Statistics obtained through nested one-way ANOVA with Tukey's multiple comparison test.
Figure 1C Top: Schematic illustration of the miR-17-92 cluster with colours marking miRNAs belonging to the same miRNA family. Bottom: TaqMan analysis of the mature miR-17-92 miRNAs in Srsf3 knockout (KO) and control (Ctrl) iPSCs (*P<0.05, ***P<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).
Figure 2B TaqMan analysis of the six miR-17-92 miRNAs in HEK293T cells overexpressing the WT-miR-17-92, 17/20a-∆CNNC or total-∆CNNC construct (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, One-way ANOVA, mean as ± SEM, n=4, biological replicates).C Western blot analysis of HA-tagged SRSF3-FL or SRSF3-∆RS expression in LIM1215 cells (detection with a HA-antibody).D TaqMan analysis of the six miR-17-92 miRNAs in LIM1215 cells overexpressing empty pCGN vector, SRSF3-FL or SRSF3-∆RS constructs (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).
Figure 3B Visualization and comparison of WT miR-17-92 SHAPE reactivities in control and SRSF3-KD cells. First panel: Mean control (blue) and SRSF3-KD (red) SHAPE reactivities, calculated over 11nt sliding windows. Second panel: ∆SHAPE reactivity differences between control and SRSF3-KD. Regions with higher reactivity in control are marked with blue shading and regions with higher reactivity in SRSF3-KD with red shading. Third panel: The SRSF3-KD to control reactivity ratios. The black boxes mark the CNNC sites.G Visualization and comparison of total-∆CNNC miR-17-92 SHAPE reactivities in control and SRSF3-KD cells. First panel: Mean control (grey) and SRSF3-KD (orange) SHAPE reactivities, calculated over 11nt sliding windows. Second panel: ∆SHAPE reactivity differences between control and SRSF3-KD. Regions with higher reactivity in control are marked with grey shading and regions with higher reactivity in SRSF3-KD with orange shading. Third panel: The SRSF3-KD to control reactivity ratios. The red boxes mark the mutated CNNC sites.