EMBO/SourceData huggingface dataset:
Figure 1A microfluidic device for quantitative recovery time measurements. Each PDMS-fabricated chip consists of six independent channels, one of which is shown, from top view (top row). Green circles indicate media inflow; black circle indicates outflow. Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers (gray circles), the height of which ensures that bacteria are imaged in a monolayer (bottom row). Growth conditions (e.g. antibiotic dose profile) are controlled via programmable syringe pumps. Fluorescent images (bottom row) show representative growth of a monolayer of E. coli BW25113 cells constitutively expressing GFP over four hours.Representative time series fluorescence data showing dose-dependent population recovery in response to transient streptomycin treatment. Here, time zero corresponds to the end of treatment (120 minutes). Trajectories show mean and standard deviation for five replicates; colors indicate increasing antibiotic concentration (2, 4, 6, 8, 10, 12 μg/mL). Fluorescence values are normalized to those at time zero. Dotted line indicates a 2-fold increase, corresponding to the recovery time for each population.Recovery time increases with total antibiotic exposure. Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations (as in panel C). When plotted against total antibiotic exposure (calculated as ), all recovery time values collapse onto a single exponential function (black line, R2 = 0.83). Recovery time was measured in response to streptomycin concencentrations of 0, 2, 4, 6, 8, 10, and 12 μg/mL, and treatment durations 30, 60, 90, and 120 minutes.
Figure 3Streptomycin treatment (closed data points, R2 = 0.83) results in significantly longer recovery times than chloramphenicol treatment (open data points). Here, the difference between two responses is statistically significant (p<0.05 by ANOVA).The addition of efflux pump inhibitor (CCCP) (closed data points, R2 = 0.91) increases population recovery time in response to chloramphenicol treatment (open data points, R2 = 0.80). Here, CCCP was added at subinhibitory concentrations (3μg/mL); in the absence of antibiotic treatment, CCCP alone did not inhibit population recovery. The CCCP-mediated increase in recovery time is statistically significant (p < 0.01).The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0.01% w/v (closed data points) to 0.05% w/v (open data points). Faster synthesis resulted in lower recovery times (p<0.001) in response to streptomycin treatment. In each of these panels, the color scheme indicates increasing antibiotic concentration, as in Figure 1C.
Figure 4Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop. Antibiotics are as follows: penicillin G (PenG), spectinomycin (Spec), gentamicin (Gent), streptomycin (Str), chloramphenicol (Cm), tetracyclin (Tet), and carbenicillin (Carb). Antibiotic concentrations used have been scaled by their respective IC50 values (solid points and open circles show response with no efflux inhibition and 2μg/mL CCCP, respectively). Colors correspond to the motifs of action shown in Figure 4A.
f1a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ-Flag-LC3 in normal medium or Earle's balanced salt solution (EBSS) for 2 h. Asterisks mark centriolar satellite proteins.b-d, Co-immunoprecipitation (IP) of OFD1 with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, haemagglutinin.e, Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown (KD) HEK293T cells.f, Co-immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3.g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation (SS). Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.
f2a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker γ-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 hserum starvation (SS).b, Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5+/+ and Atg5−/− MEFs subjected to 24 hserum starvation. a, b, Data shown represent mean ± s.d. percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments.
f3a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate samples. ***P  0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments. Un, untreated; SS, serum-starved.
f4a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with alpha-tubulin.b, Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia for 500 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test.c, Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation.d, e, Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. Data shown represent 100 cells per well in triplicate samples.d, e, Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation. Data shown represent 100 cells per well in triplicate samples.f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.
Figure 1 (A) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity. Triangles point to immune cells (clusters), including some neutrophils known to express PI3Kγ highly. In the negative control, the primary antibody was replaced by an equal volume buffer. The number of included patients, gender, and diagnosis are summarized  (B) PI3Kγ expression in human primary hepatocytes from 20 male (♂) or female (♀) donor pools (HEP, DP20), but not non-parenchymal cells (NPC). Isolated human leukocytes from healthy volunteers (LEU) served as positive controls. The characterization of the donor pools is summarized   (D) Survival of WT, PI3Kγ null (left) and liver-specific PI3Kγ knockout mice (PI3Kγ floxtg/tg x AlbCre(tg)/tg, middle) or systemic application of the PI3Kγ inhibitor, AS605240 (right) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension. Animal numbers: B6: 86 (%male: 52, %female: 48), B6-Pi3kcgko/ko: 59 (%male: 51, %female 49), B6-Pi3kcgflox/flox x AlbCre(tg)/tg: 25 (%male: 68, %female 32), FVB/N Vehicle: 25 (%male: 44, %female 56), FVB/N AS: 43 (%male: 44, %female 56)
Figure 2 (C) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples.  (F) T-LipoAS effectively inhibits fMLP-stimulated PI3Kγ, reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes. The mean pAKT/AKT ratio and standard deviation (SD) were calculated for all experiments separately.  (G) Episcopic imaging of mice treated with T-LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY-635 in the different compartments.
Figure 3 (B Pro- anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).  C) anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).  (F,G) Concentration of (F) neutrophils and (G) monocytes in 2 mL peritoneal lavage.
Figure 4 (A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS).
Figure 1(A) GFP-PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation (GS) for 4 h. The nuclei were stained with Hoechst 33342. Scale bar: 10 μm.(B) Immunoblotting (IB) analysis of wild type (WT) and PAQR3-deficient MEFs after GS for different time as indicated.(C) WT and PAQR3-deficient MEFs were treated with 80 mM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole-cell lysates were harvested for IB analysis of LC3-II.(D) Autophagosome-like structures in WT and PAQR3-deleted MEFs were detected after GS for 4 h by transmission electron microscopy. * indicates autophagosome-like structures. N, nucleus.(E) Quantitative analysis of autophagic vacuoles in transmission electron microscopy images. Thirty cells were quantified from each independent experiment，which was repeated for three times with similar results. Values are presented as mean ± SD, ***p < 0.001.(F) Immunofluorescence staining of LC3 in WT or PAQR3-deficient MEFs after GS for 4 h. The arrowhead indicates representative LC3-positive puncta. Scale bar: 10 μm.(G, H) HeLa cells were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control virus. After GS for 1 h, the cell lysates were harvested for IB.
Figure 2(A~C) HeLa cells stably expressing GFP-DFCP1 were transfected with or without PAQR3-specific shRNA. GFP signals were observed in these cells before or after glucose starvation (GS, shown in A). The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP-positive puncta per cell is illustrated in C. Values are presented as mean ± SD, *** P < 0.001. Scale bar: 10 μm.(D) GFP-WIPI1 was transfected into WT or PAQR3-deficient MEFs. 24 h later, the cells were incubated under GS for 4 h, followed by fluorescence observation. Scale bar: 10 μm.(E) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium (NM) or GS. Then PI(3)P level was determined by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L or UVRAG used in the assay (n = 5; ***p < 0.001; ns, not significant).(F) HeLa cells infected with control or PAQR3-overexpressed lentivirus were treated with GS for 4 h. PI(3)P levels were determined as in Figure 2E(n = 5; **p < 0.01; ns, not significant).(G, H) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants. At 24 h after the transfection, the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody, followed by immunoblotting. The activity of ATG14L-linked VPS34 activity was detected by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; **p < 0.01).
Figure 3(A) Interaction between endogenous PAQR3 and the components of VPS34 complex in HEK293T cells. The cell lysates were used for immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.(B) Cytosol and Golgi fractionations were isolated from the livers of PAQR3-deleted mice and their littermate controls. Equal protein amounts of cytosol and Golgi fractions were subjected to IB.(C) HEK293T cells were transiently transfected with Myc-tagged PAQR3. At 24 h after transfection, the cells were treated with normal medium (NM)，amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h respectively. The cell lysates were then used in IP and IB.(D) Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM, AS or GS conditions. The relative abundance of VPS34-binding partners was determined by IB. Each IP was normalized to the amount of VPS34.(E) HEK293T cells were transfected with the plasmids as indicated and the cell lysates were used in IP and IB.(F) The lysates of WT or PAQR3 knockout MEFs were used in IP and IB.(G) MEFs were infected with control or PAQR3-expressing lentivirus. Then a quantitative immunodepletion assay was performed with increasing amounts of the indicated antibodies. Supernatants of MEF lysates after immunodepletion (Post-IP) were examined to determine the level of VPS34 complex proteins.
Figure 4(A) A two-step co-IP assay in HEK293T cells to determine the ternary complex containing PAQR3, Beclin1 and ATG14L. The procedures of the two-step co-IP are outlined in the top. The samples were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.(B) HeLa cells were transiently transfected with the plasmids as indicated. 24 h after transfection, the cells were treated with or without glucose starvation (GS) for 4 h. The cell lysates were used in IP and IB.(C, D) Different truncations of ATG14L (C) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.(C, D) Different truncations of Beclin1 (D) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.(E) Purified GST or GST-fused PAQR3 (1-71) (GST fused NH2-terminal 71aa of PAQR3) on glutathione agarose beads was mixed with purified His tagged proteins as indicated. After incubation at 4 °C for 3 h, the samples were washed extensively and subjected to IB.(F, G) Different PAQR3 deletion constructs were co-transfected with Flag-tagged Beclin1 (F) into HEK293T cells. The cells were then subjected to IP and IB.(F, G) Different PAQR3 deletion constructs were co-transfected with GFP-fused ATG14L (G) into HEK293T cells. The cells were then subjected to IP and IB.
Figure 5(A) HeLa cells were cultured in normal medium (NM) or glucose starvation (GS) for 4 h. The starved lysates were treated with calf intestinal phosphatase (CIP) to remove phosphate groups and analyzed by immunoblotting (IB) with Phos-tag gel or regular SDS-PAGE.(B) The cell lysates from HeLa cells were collected and analyzed after GS for indicated time (lane 1~5), or GS for 2 h followed by 2 h chase in NM (lane 6). The cell lysates were subjected to Phos-tag gel and regular SDS PAGE respectively.(C) WT or AMPKa1/2 double knockout MEFs were incubated under NM or GS for 4 h, followed by IB with Phos-tag gel or regular SDS-PAGE.(D) HeLa cells were incubated with compound C (20 mM, 30 min) with or without GS, followed by IB with Phos-tag gel or regular SDS-PAGE.(E) Bacterial purified His-tagged NH2-terminal 71 amino acid of PAQR3 was incubated with or without purified Flag-tagged ATG14L. Then the complexes were treated with WT AMPK or DN-AMPK (dominant negative AMPK) for the indicated time in vitro. PAQR3 phosphorylation was examined by Phos-tag gel.(F) HeLa cells infected with control or ATG14L-expressing lentivirus were incubated under GS for different time as indicated. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.(G) HeLa cells were infected by lentivirus expressing control shRNA or ATG14L-specific shRNA. Then the ATG14L knockdown cells were stably re-expressed with shRNA-resistant ATG14L (rATG14L) or Beclin1 and incubated under NM or GS for 4 h. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.
Figure 6(A) Bacterial purified His-tagged WT or T32A NH2-terminal 71aa of PAQR3 was incubated with or without Flag-tagged ATG14L purified from HEK293T cells. Then the complexes were incubated with AMPK for the indicated time and subjected to Phos-tag gel or regular SDS-PAGE.(B) PAQR3-deficient HeLa cells were transfected with WT or T32A mutants of PAQR3 plasmids. 24 h later, the cells were incubated with normal medium (NM) or glucose starvation (GS) for 4 h, followed by immunoblotting (IB) in Phos-tag gel or regular SDS-PAGE.(C) HeLa Cells were incubated with NM or GS for 1 h or treated with compound C (C.C, 20 mM, 30 min) prior to GS for 1 h. In parallel, 25 mM 2-DG or 1 mM AICAR was added in NM for 1 h. Cell lysates were analyzed by IB.(D) HEK293T cells were transfected with Myc-tagged PAQR3. 24 h after transfection, the cells were treated with GS for 4 h. Then the cell lysates were used in IB and immunoprecipitation (IP) with the antibodies as indicated. The immunoprecipitates were treated with CIP to remove phosphate groups and analyzed by IB in Phos-tag gel or regular SDS-PAGE.(E) WT, phosphorylation defective (T32A), or phospho-mimetic (T32E) PAQR3 were transfected in the presence or absence of constitutively active AMPK (CA-AMPK) into PAQR3-deficient HeLa cells as indicated. 24 h later, the cells without AMPK transfection were treated with GS for 4 h. 10% of the cell lysates were subjected to IB to detect PAQR3 expression level (shown in Appendix Fig S4A), the remaining cell lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody, followed by VPS34 activity measurement by a quantitative PI(3)P ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; *p < 0.05; **p < 0.01 as compared to the first group).(F) PAQR3-deficient HeLa cells were infected with lentivirus expressing WT or T32A PAQR3. Then PAQR3 expression levels were examined by IB.(G) Both WT HeLa cells and PAQR3 knockout HeLa cells infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively. Cell lysates were analyzed by IB.
Figure 7(A, B) Representative immunohistochemical images of LC3 staining in skeletal muscle (A) and liver (B) after 80 min exercise in WT and PAQR3-deleted mice. Scale bar in (A): 20 μm. Scale bar in (B): 50 μm.(C, D) Immunoblotting analysis of skeletal muscle (C) and liver (D) from WT or PAQR3 knockout mice at rest or after 80 min exercise.(E) Abnormal limb-clasping of PAQR3 knockout mice when suspended by its tail. Quantification scoring of the limb clasping phenotype is shown on the right (n = 7 for each group; **p < 0.01).(F) Motor performance was measured using an accelerating rotarod apparatus, and the time of each mouse spent on the rotating rod until it fell off was recorded. Average time on the rod for each group is shown (n = 7 for each group; **p < 0.01).(G) Co-immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype. Immunoprecipitation (IP) and immunoblotting (IB) were performed using the antibodies as indicated.(H) The supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration analysis, followed by IB.(I) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI(3)P ELISA assay (n = 5; **p < 0.01).
Figure 1A. Labeling of non-permeabilized (top) and permeabilized (bottom) cells demonstrate that surface labeling does not label internal proteins. GluA2 labeling (green) is visible both on the surface and internally, while DARPP32 labeling (red) is only visible internally.B. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no treatment or 20 min 2APB and dantrolene. Bottom. Quantified expression of GluA2 under both conditions, n = 9 replicates of 10 cells each, Student's t test.C. Representative images of surface GluA1 and DARPP32 expression in striatal neurons from co-cultures with either no treatment, 30 min DHPG, or 2APB and dantrolene pretreatment followed by DHPG. D. Quantified expression of GluA1 under each condition, n = 9 replicates of 10 cells, p < 0.001, ANOVA. E. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. F. Quantified expression of GluA2 under each condition, n = 12 replicates of 10 cells each, p < 0.001, ANOVA. G. Representative images of surface myc-GluA2, internal myc-GluA2, and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. H. Quantified expression of surface myc:total myc under each condition, n = 4 replicates of 10 cells each, p < 0.05, ANOVA. All error bars represent s.e.m. Abbr. A1 = GluA1, A2 = GluA2, D = DARPP32, s-myc = surface myc, i-myc = internal myc, m = merge.
Figure 2A. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG) or for 30 min with DHPG and pretreated with Xestospungin C and S107 (XeC/S107 + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 or 20 cells each, p < 0.0001, Student's t test.B. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG), or for 30 min with DHPG and pretreated with S107 (S107 + DHPG), or for 30 min with DHPG and pretreated with Xestospungin C (XeC + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, ANOVA.C. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under control or 10 min Thapsigargin (TG) treatment. Bottom. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. D. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with treatment with either no DHPG or with DHPG for 30 min, after 15 min of Thapsigargin. Bottom. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, Student's t test. E. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min, after 5 min of BAPTA. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, p < 0.001, Student's t test. F. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA-AM. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.