EMBO/SourceData huggingface dataset:
Figure 4(A) Indicated strains were spotted 5-fold dilutions on SC plates in 3 biological replicates and 2 technical replicates (n=5), and plates were incubated at room temperature, 30oC, and 37oC. Upper panel: WT, dfm1∆, orm1∆, and dfm1∆orm1∆ were compared for growth by dilution assay. Middle panel: WT, dfm1∆, orm2∆, and orm2∆tsc3∆ were compared for growth by dilution assay. Bottom panel: WT, dfm1∆, orm1∆, orm2∆, and orm2∆, and orm1∆orm2∆ were compared for growth by dilution assay. Upper, middle, and lower panel: gray arrowhead depicts growth phenotype of dfm1∆orm1∆, dfm1∆orm2∆, and orm1∆orm2∆ respectively. (B) dfm1∆orm1∆ confers resistance to myriocin and sensitivity to PHS. WT, dfm1∆, orm1∆, and dfm1∆orm1∆ strains were grown to log-phase in SC medium, and 5-fold serial dilutions of cultures were spotted on YPD plates containing either drug vehicle alone, 1 mM of myriocin and 10 μM of PHS with each condition performed in 3 biological replicates and 2 technical replicates (n=5). Plates were incubated at room temperature and photographed after 3 days. Open circle indicates growth phenotype of dfm1∆orm1∆ cells.
Figure 5(A) Degradation of Orm2 depends on Dfm1 and not Der1. The indicated strains expressing Orm2-RFP were grown into log phase and degradation was measured by cycloheximide chase (CHX). After CHX addition, cells were lysed at the indicated times, and analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates (n=3). (B) Same as (A) except degradation of Orm2-RFP was measured in WT and der1∆ cells (3 biological replicates; n=3).(D) Dfm1's SHP box is not required for degradation of Orm2-RFP. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3).(G) Dfm1's WR motif, GxxxG motif, substrate binding and lipid thinning function are required for degradation of Orm2-RFP. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3).(H) Dfm1 L1 residues are required for binding to Orm2. Orm2-RFP and binding to retrotranslocation-deficient Dfm1 L1 mutants was analyzed by co-IP. The IP was analyzed for presence of Dfm1-HA. As a negative control, cells not expressing Orm2-RFP were used (3 biological replicates; n=3). Band intensities for all western blots were normalized to PGK1 loading control and quantified by ImageJ.
Figure 6(A) E3 ligase Tul1 is required for Orm2 degradation. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.(B) Cdc48 and the proteasome are required for Orm2 degradation. Same as (A), except cdc48-2 and hrd2-1 were analyzed for Orm2-RFP degradation (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.(C) Dfm1 does not function in the post-ubiquitination step of Orm2 degradation pathway. Indicated strains expressing Orm2-RFP were grown into log phase. Cells were lysed, and microsomes were collected and immunoprecipitated with α-RFP conjugated to agarose beads. Samples were then subjected to SDS-PAGE and immunoblot by α-Ubiquitin and α-RFP (3 biological replicates; n=3).(D) ERAD mutants do not rescue temperature-sensitive lethality of tsc3∆. Indicated strains were grown to log-phase in SC and serially diluted cultures were plated on SC plates and incubated at 37°C and imaged at Day 2 (3 biological replicates, 2 technical replicates; n=5).
Figure 7(A) Phosphorylated Orm2 accumulates in the absence of Dfm1. Phos-tag western blot analysis shows that there is an accumulation of phosphorylated Orm2 in dfm1∆ cells. The indicated strains were grown to log phase, treated with vehicle or 1.5 μM myriocin for 1 hour and subjected to SDS-PAGE or Phos-tag western blot analysis via blotting for Orm2 with α-RFP and PGK1 with α-PGK1 antibodies (2 biological replicates; n=2).(C) dfm1∆ cells block the degradation of phosphorylated mimic of Orm2 (Orm2-3D). In the indicated strains, degradation of Orm2-3A-GFP and Orm2-3D-GFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted α-GFP (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.
Figure 8(A) Dfm1-HA binding to Orm2-GFP, Orm2-3A-GFP, Orm2-3D-GFP, Orm1, Lcb1, and Lcb2 were analyzed by co-IP. As negative control, cells not expressing Dfm1-HA were used. Also, Sec61 was also included to test for non-specific binding (3 biological replicates; n=3). (B) Same as (A), except co-IP was performed on WT and orm2∆ cells and Dfm1-HA binding to Orm1, Lcb1, and Lcb2 was analyzed (3 biological replicates; n=3). (C) Same as (A), except Orm2-GFP binding to Dfm1-HA, Orm1, Lcb1, and Lcb2 was analyzed by co-IP (3 biological replicates; n=3).
Figure 1(A) Cellulase functional screening by Congo Red assay. Halos on the plate indicate cellulase activity. Pc: Pseudomonas cellulosa, Bs: Bacillus subtilis. Numbered strains are from the C. elegans native gut microbiome(B) Production of reducing sugars by cellulose-degrading bacterial strains. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.(D) Colony-forming units (CFU) of various bacterial species in the worm gut. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.(E) C. elegans colonized with cellulolytic P. cellulosa and incubated with different carbon sources. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.
Figure 2(A) Incorporation of 14C-labelled cellulose. Worms were first colonized with cellulolytic bacteria and then incubated with 14C-cellulose to allow carbon utilization. Antibiotic treatment was applied before 14C measurement of worms to eliminate bacterial interference in isotopic reading. 14C-cellulose incorporation measurement reflected the isotopic carbon incorporation in C. elegans. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In A, experiments were performed in biological triplicates using 200 worms per data point. In A error bars represent 95% confidence intervals for the mean.(B) Induction of the worm fat-7p::GFP metabolic reporter. Adult worms pre-colonized with P. cellulosa were incubated in liquid media for 24 h with the indicated carbon sources, and GFP fluorescence was read in individual worms on a BioSorter large object sorter. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In B, error bars represent 95% confidence intervals for the mean. In B, each point represents a different worm in biological replicates, n=96, n=53, and n=80 from left to right.(C) Effect of nutrition on larval yield. Pre-colonized worms were separated into the individual wells of a 384-well plate, and larvae counts in each well were read after 48 h. Larval yield comparison indicates the nutritional benefit of cellulolytic activity. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In C, the central band represents the median, notches represent 95% confidence intervals for the median, the box edges represent the inter quartile range (IQR) and the whiskers extend an additional 1.5 IQR beyond the boxes. In C, each point represents a different worm in biological replicates, n=74, n=79, n=79, n=89, n=88, n=86 from left to right.
Figure 3Synchronized adult worms were colonized for two days on plates containing E. coli OP50 + P. cellulosa (Pc), E. coli OP50 + Lp, or E. coli OP50 + Pc + Lp. (B) After 36 h outgrowth of the pathogen, worms pre-colonized with P. cellulosa in the presence of L. plantarum (E. coli OP50 + Pc + Lp) showed lower pathogen burden than P. cellulosa monocolonized worms when CMC was used as the carbon source. Data shown are fold change Salmonella infection (CFU/worm) relative to the P. cellulosa-only condition. Error bars represent 95% confidence interval of the mean. CMC = 0.01% carboxymethylcellulose (w/v). All experiments were performed in biological triplicates. Student's t test was used for statistical analysis.
Figure 1Average fluorescence signals aligned to the onset of scratching trains across the GCaMP6s group (red line) and EYFP group (blue line) in C48/80 model (C). Heatmap illustration of fluorescence changes during each corresponding scratching train in GCaMP6s group. n = 8 mice/group. 2 trails for each are shown except for 1 mouse where 1 trial was performed (D). *** P < 0.001. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group.J, Effect of chemogenetic inhibition (J) of dopaminergicA11-SDH neurons on scratching behaviors in response to C48/80 (left) and CQ (right) stimuli. ** P < 0.01, *** P < 0.001. Two-way ANOVA followed by Bonferroni's test. n = 6-8 mice/group.
Figure 2Average fluorescence signals aligned to the onset of scratching trains across the GCaMP6s group (red line) and EYFP group (blue line) in DCP model (B).F, Effect of chemogenetic inhibition (F) of dopaminergicA11-SDH neurons on DCP-induced scratching. * P < 0.05. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.G Effect of chemogenetic activation (G) of dopaminergicA11-SDH neurons on DCP-induced scratching. * P < 0.05. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.
Figure 3A Effect of SCH23390 and sulpiride on scratching behaviors induced by C48/80 (left) and CQ (right). *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 7-8 mice/group.C Effect of SCH23390 and sulpiride on DCP-induced scratching behaviors. * P < 0.05. One-way ANOVA followed by Bonferroni's test. n = 8 mice/group.D The Drd1 mRNA level in the spinal cord after DCP or vehicle treatment. * P < 0.05. Student's t-test. n = 8 mice/group.G Statistical curve shows the firing frequency evoked by step current injection into DRD1+ neurons that incubated with vehicle or SKF38393. *** P < 0.001. Two-way ANOVA. n = 8 neurons/group.I Statistical shows the resting membrane potential in DRD1+ neurons incubated with vehicle or SKF38393. * P < 0.05. Paired Student's t-test. n = 8 neurons/group.K Representative traces from one spinal DRD1+ neuron show that blue light stimuli did not induce spontaneous firing (holding potential, -65 mV, lower panel) but increased the existed firing of DRD1+ neuron (holding potential, -60 mV, upper panel). Arrows indicate blue light stimuli (2-3 mW/cm2, 5 pulses, 2 ms duration, 20 Hz, repeated every 1 s). shows the change of firing frequency from individual DRD1+ neurons after blue light stimuli. ** P < 0.01. Paired Student's t-test. n = 8 neurons/group. Data information: Bars represent mean values. Error bars indicate the SEM.
Figure 4G Effect of chemogenetic activation of spinal DRD1+ neurons on spontaneous itch behaviors. *** P < 0.001. Student's t-test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.I Effect of chemogenetic inhibition (left) or activation (right) of spinal DRD1+ neurons on the DCP-induced chronic itch behaviors. * P < 0.05, ** P < 0.01. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.
Figure 5F-H Representative images show non-overlap of tdTomato with EGFP in the SDH of Drd1-tdTomato;Drd2-EGFP mice. Scale bar, 100 μm.I Representative image shows the co-localization of NeuN with tdTomato. Scale bar, 50 μm. Inset scale bar, 25 μm.J-M Representative images show the co-localization of NK1R (J), CGRP (K), IB4 (L), and PKCγ (M) with tdTomato. Scale bar, 100 μm.N Co-localization of tdTomato fluorescence with vGluT2-probe. Scale bar, 50 μm. O FISH double-staining of Drd1-probe with vGluT2-probe. White arrows and arrowheads show double-labeled neurons and vGluT2 single-labeled neurons, respectively. Scale bar, 10 μm. P Staining of PAX2 in Drd1-tdTomato mice. Scale bar, 50 μm. Q Fluorescence of tdTomato and EGFP in the SDH of Drd1-tdTomato;Gad67-EGFP mice. Scale bar, 50 μm.
Figure 6C Representative images show the reconstitution of pre- and post-mGRASP to form an intact GFP (green), which mapped synaptic connectivity between spinal DRD1+ neurons (red, as presynaptic constructs) and GRPR+ neurons (upper panels). High-magnification images in the white frames were shown in lower panels. Scale bar, 20 μm in upper panel and 10 μm in lower panel.G Representative traces of the whole-cell voltage-clamp show the blue light (470 nm, 2 ms)-evoked EPSCs recorded in a GRPR+ neuron before (black) and after 20 μM DNQX perfusion (gray). H Representative traces show the effect of TTX alone (purple) and the combined presence of 4-AP (gray) superfusion on the blue light-evoked EPSCs (black) recorded in a GRPR+ neuron.I Representative traces show the effect of TTX + 4-AP (gray) on the blue light (470 nm, 2 ms)-evoked EPSCs (black) recorded in a GRPR+ neuron.
Figure 7D Single-cell RT-PCR shows the expression of Drd1, Grp, NeuN, and Gapdh mRNA in tdTomato+ neurons. N, negative control. * indicate Drd1+Grp+ neurons.I Effect of DNQX, RC-3095, or vehicle on the spontaneous scratching of mice with spinal DRD1+ neurons expressing mCherry or hM3Dq and activated by CNO. ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.J Effect of DNQX, RC-3095, or vehicle on the scratching behaviors of mice with spinal DRD1+ neurons expressing mCherry or hM3Dq and activated by CNO in C48/80 model (left) or CQ model (right). * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 6 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.
Figure 1(C) Enrichment of proteins detected by mass spectrometry on a pool of TATA-box promoters over control DNA sequences(D) over a pool of DRE promoters. 3 biological replicates were performed for each promoter and control pool, significance measured with a Limma p-value<0.05.
Figure 2D. Western blots of the DNA-bound fraction eluted off the beads in a DNA-affinity purification assay. Super core promoter 1 (SCP1) was used a positive control to bind TFIIA-β-FLAG (top panel). DRE promoter pools of varying lengths were assayed for their ability to bind TFIIA. Sonicated salmon sperm DNA was used as competitor DNA and titrated from 100ng to 1.6ug per reaction. Histone H3 was used as an abundant non-specific DNA interacting protein for loading control. From top to bottom we have used promoter fragments ranging at 121bp, 350bp and 1kb in length.E. ChIP-seq signal of GTFs and select housekeeping promoter binders from Drosophila S2 cells and embryos normalized to nascent transcription level as measured by PRO-seq at the respective promoter types.
Figure 3B. Western blot on FLAG tagged TBP and DREF 0,1 and 3 hours after auxin addition showing protein degradation.E. Fisher's exact test for motif enrichment in TBP and DREF downregulated promoters compared to all expressed promoters. Log2 of the Odds ratio displayed. The dashed line are set at a value of 1 and -1.
Figure 4E. ChIP-seq coverage (input normalized) of TBP and TRF2 at TBP and/or TRF2 dependent promoters, and all other active promoters ('unchanged'). Orange: TBP dependent promoters (i.e. promoters downregulated upon TBP depletion); blue: TRF2 dependent promoters; purple: promoters downregulated when both, TBP and Trf2 are depleted (orange, blue and purple are not mutually exclusive sets of genes); white: unaffected promoters. Boxes represent the upper and lower quartiles, with the middle band at the median. The whiskers represent the upper and lower 5th percentiles. Data was taken from two merged biological replicates.F. Western blot of anti-FLAG antibody of TBP-AID, Trf2-AID and a double tagged TBP-AID + Trf2-AID cell lines from a multi-day time course of auxin treatment showing prolonged depletion.J. qPCR on an auxin treatment time course of TBP and Trf2 dependent genes upon individual depletion of TBP or Trf2 and a double depletion of both. Error bars represent the standard deviation across three biological replicates.
Figure 5C. PRO-seq signal at all expressed promoters, represented according to their motif content in box plots. Boxes represent the upper and lower quartiles, with the middle band at the median. The whiskers represent the upper and lower 5th percentiles across two biological replicates.E. Luciferase assay in which Gal4 DNA binding domain fusion proteins were recruited to 4xUAS sites upstream of a minimal housekeeping Rps12 promoter. Measurements are normalized to Renilla luciferase (transfection control) and GFP. * denotes proteins activating a housekeeping promoter with a log2FC>1.5 and p-value<0.05, two-tailed student's t-test. Error bars represent standard deviation across four biological replices.
Figure 6A. Distribution of CAGE signal from mixed D. mel embryos (0-24h) centered on the location of promoter DNA motif sequence set at position 0 across the 6 main promoter types investigated in this study.D. Box plot of the percent of STAP-seq signal (i.e. percent of initiation) originating at the dominant TSS at core promoters (CPs; N = 1266) and random regions (N = 639) that are activated to similar extent by both GFZF and MED25 recruitment. Cofactor recruitment STAP-seq data from (Haberle et al, 2019), three independent biological replicates merged. *P≤0.01; Wilcoxon rank sum test.
Figure 1C-F (C, E) Immunofluorescence labelling with an antibody that recognizes both full-length and cleaved Xkr8 in developing (P0, P8) and adult S1 cortex and hippocampus. (D, F) Immunofluorescence labelling with an antibody recognizing full-length uncleaved Xkr8 in developing (P0, P8) and adult S1 cortex and hippocampus. Loss of Xkr8 immunosignal in double transgenic Xkr8flx/flx;Emx1::Cre animals (Xkr8 cKO) is observed with both Xkr8 antibodies (C-F).
Figure 2A, B Organotypic slices of Xkr8 WT and cKO hippocampus with Thy1::GFP neurons (green) were labelled with PtdSer-binding fluorescently tagged Annexin V (magenta) at 16-19 DIV. The fluorescence of bound Annexin V was quantified on ImageJ by measuring the integrated density of fluorescent structures within the same volume for each slice and expressing the value as fluorescence units (f.u.; two-tailed Student's t-test, each dot represents an individual slice preparation, n = 4).C Immunofluorescence labelling of active caspase-3 in developing brain from P8 to P28 in Xkr8 WT and Xkr8 cKO brains was quantified and compared by nested design and mixed model ANOVA (F(2,30) = 0.86, p = 0.433), each dot represents an individual mouse (n = 6 per age group)
Figure 3A-C The size and density of axonal boutons on Thy1::GFP axons (green) in Xkr8 WT and cKO animals (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 6 per age and genotype group).D, E Dendritic spine morphology (D) and density (E) of Thy1::GFP+ CA1 neurons in Xkr8 WT and cKO animals from P8 to P28 (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 5-6 per age and genotype group).J-L The density of cortical (vGluT1+, J) and thalamic (vGluT2+, K) and the ratio of vGluT1+ and vGluT2+ inputs (L) in Xkr8 WT and cKO brains (nested design and mixed model ANOVA, each dot represents an individual mouse, n = 6 per age and genotype group).
Figure 4A-F The immunolabelling of internalized pan-axonal material (A, B), pre-synaptic vGluT1 particles (C, D) and post-synaptic PSD95 particles (E, F) within Iba1+ microglia in P8 cortex of Xkr8 WT and Xkr8 cKO mice was quantified per individual 3D reconstructed microglial cell. The total volume of internalized particles was normalized to microglial cell volume and compared by nested design and mixed model ANOVA, each dot represents an individual mouse (n = 3 per genotype group). Data presented as mean ± SEM; ** p < 0.01, **** p < 0.0001; scale bar 10 µm, grid cell 10 µm.
Figure 5A, B Corticospinal axons of the axonal bundles of pyramidal tracts in medulla of P8 and P28 Xkr8 WT and cKO mice were visualized by Palmgren staining and quantified per each bundle, delineated by a dashed line (Mann-Whitney test, n = 6 mice per genotype, the data are presented as median and quartiles).To label callosal projections in vivo, cholera toxin subunit B (CTB) was injected stereotactically into corpus callosum of Xkr8 WT and cKO brains at P30-32. Back-labelled neurons were quantified in L4 of primary (S1) and secondary (S2) somatosensory cortex.
Figure 6Xkr8 cKO neurons had increased mean sEPSC amplitude Data were analyzed by two-tailed Student's t-test ; n = 7-14 neurons per genotype.F-H Evoked excitatory postsynaptic currents (eEPSC) of Xkr8 cKO neurons showed a trend to decrease (p = 0.08) (F, G) and the ratio of eEPSC and sEPSC amplitudes was lower in Xkr8 knockout neurons (H). two-way repeated measures ANOVA (H); n = 7-14 neurons per genotype.
Figure 7A Brain regions with significantly increased functional connectivity in Xkr8 cKO mice compared to Xkr8 WT mice as identified by seed-based mapping of rsfMRI networks (t-test, p < 0.05 FWE cluster-corrected, with cluster-defining threshold of t24 > 2.07, p < 0.05; Colored regions represent the areas of the brain exhibiting increased functional connectivity in Xkr8 cKO compared to Xkr8 WT brains; color scale represents the level of functional correlation; abbreviations specify anatomical designations of identified regions. ACA: anterior cingulate cortex, AI: agranular insular area, AUD: auditory areas MOp: primary motor area, MOs: secondary motor area, PL: prelimbic area, SSp: primary somatosensory area, STR: striatum, STRd: striatum dorsal region, SUB: subiculum, TH: thalamus, VIS: visual areas, VISC: visceral area.
Figure 1A. EGFR (pY1068, left), Akt (pS473, middle) and Erk (pT202 and pY204, right) phosphorylation response in WT (red) compared to p22phox-KO (green) MCF7 cells as function of EGF concentration (ng/ml; nM) upon 5' stimulation with different doses of EGF-Alexa647 quantified from Western blot analysis. N=4 biological replicates with mean±SD, P:unpaired two-tailed t-test. B. Same as (A) comparing WT (red) to RPTPγ-KO (blue) MCF7 cells. N=3 biological replicates with mean±SD, P: unpaired two-tailed t-test. C. Quantitative Western blot analysis as in (A) comparing WT (red) and RPTPγ-KO (blue) MCF7 cells after EGF stimulus (20, 80, 160 ng/ml from (B), left column: w/o gef.) to the cells from the corresponding cell line treated with 10 μM of the EGFR-inhibitor Gefitinib for 1 h and the indicated EGF concentration for the last 5' (ng/ml). N=3 biological replicates with mean±SD, P: unpaired two-tailed t-test. D.E. Comparison of normalized EGFR- (pY1068-) phosphorylation as a function of EGF concentration (N=10, from Figs 1A, 1B, EV1E, red) to EGF-Alexa647 bound to WT MCF7 cells at the corresponding, indicated concentrations normalized to the 160 ng/mL, measured by fluorescence microscopy. N=5 biological replicates, n=16-19 fields of view, mean±SD, P: One-way ANOVA with Tukey's multiple comparison test.