EMBO/soda-nel huggingface dataset:
STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of Th17 cells (2020)
Figure 5

Figure 5. STIM1 and STIM2 in immune cells are essential for immunity to mucosal C. albicans infection.

(A-G) Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (A) Body weight (BW) measured for 7 days and normalized to BW at day 0 of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice. (B) Quantification of CFU of C. albicans per gram tongue tissue of the indicated mice at day 7 p.i. Data are mean ± SEM from 4 repeat experiments and 12 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i. Arrows and dashes lines indicate C. albicans infiltrated areas. (D) Histological images of H&E (left) and PAS (right) stained tongue tissue at day 7 p.i. Clusters of polymorphonuclear (PMN) cells (neutrophils, white arrows) and Candida (black arrows) are indicated. (E) Frequencies of Cd11b+Gr1+ neutrophils in the tongues from the indicated mice at day 7 p.i. Bar graphs represent the mean ± SEM from 3 experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice. (F,G) Cytokine production by CD4+ T cells isolated from submandibular lymph nodes of WT, Stim1fl/flVav-iCre, Stim1fl/flStim2fl/flVav-iCre and Stim1fl/flCd4Cre mice 7 days after sublingual infection with 2x107 CFU C. albicans. Representative contour plots of cytokine production (F) and percentages of CD4+ T cells producing TNF-α, IFN-γ, IL-17A, IL-17F and GM-CSF (G). Data are the mean ± SEM from two independent experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice. (H-I) Candicidal function and ROS production of neutrophils depend on SOCE. (H) Coculture of Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice with C.albicans in vitro for 0, 30, 60 and 90 min (MOI 0.5). Percentages of live C.albicans isolated from neutrophils that were lysed at the indicated time points. Data are the mean ± SEM from 6 mice per genotype and 3 independent repeat experiments. (I) ROS production by Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice was measured after loading of cells with dihydrorhodamine 123 and stimulation with 10 nM PMA for 30 minutes. Representative histogram plots and bar graphs indicating the percentages of ROS+ neutrophils normalized to WT neutrophils stimulated with PMA. Data are the mean ± SEM from 2 mice per genotype and 2 independent repeat experiments.

Data information: Statistical analysis by unpaired Student's t-test. *p<0.05, **p<0.01, ***p<0.001.
STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of Th17 cells (2020)
Figure 6

Figure 6. STIM1 is required for T cell-mediated immunity to systemic fungal infection.

(A-D) Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (A) Body weight (BW) change measured for 7 days p.i. relative to BW at the day of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT and 7 Stim1fl/flCd4Cre mice. (B) Quantification of CFU of C. albicans isolated from the tongues of mice at day 7 post-infection (p.i.). Data are mean ± SEM from 4 repeat experiments and 7-13 mice per genotype. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i. (D) Histological images of H&E (left) and PAS-stained (right) tongue tissue at day 7 p.i. Scale bars 100 μm. Arrows point to clusters of polymorphonuclear (PMN) cell (neutrophil) infiltrates.

(E-K) Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (E) Kaplan-Meier survival plot showing survival of mice. Data from 5 Stim1fl/fl Cd4Cre mice and 12 WT mice is shown. (F) Body weight (BW) of mice normalized to BW at the day of infection. Data are the mean ± SEM from 4 independent experiments and 8 WT and 7 Stim1fl/flCd4Cre mice. (G) Representative macroscopic images of kidneys at day 6 p.i. (H) CFUs of C. albicans per gram tissue isolated from the liver, the right kidney and the lung of mice at day 6 p.i. Each dot represents one mouse. (I) Representative histological images of H&E and PAS stained kidneys of mice at day 6 p.i. Magnification 6.4x (left) and 40x (right). White arrows define areas infiltrated with leukocytes (H&E) and C. albicans (PAS). (J) Frequencies of CD4+ T cells isolated from the spleens of mice at day 6 p.i. producing the indicated cytokines after restimulation with 20 nM PMA and 1 μM ionomycin for 6 hours and analyzed by flow cytometry. (K) Frequencies CD45+Cd11b+Gr-1+ neutrophils in the blood of mice at day 6 post-infection analyzed by flow cytometry. Data in J are the mean ± SEM from 2 independent experiments and 6 WT and 4 Stim1fl/flCd4Cre mice; data in K are the mean ± SEM from one experiment and 2 WT and 3 Stim1fl/flCd4Cre mice.

Data information: Statistical analyses in A, B, F, J, K by unpaired Student's t-test; in E by Log-rank (Mantel-Cox) test and in H by Mann Whitney U test. Significance levels: *p<0.05, **p<0.01.
STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of Th17 cells (2020)
Figure 7

Figure 7. STIM1 differentially regulates expression of genes associated with pathogenic and non-pathogenic Th17 cells.

RNA-seq analysis of CD4+ T cells isolated from 3 WT and 3 Stim1fl/flCd4Cre mice that were differentiated into pathogenic Th17 cells (pTh17: IL-1β, IL-6, IL-23) and non-pathogenic Th17 cells (npTh17: IL-6, TGFβ1).

(A) Differentially expressed genes (DEG) in WT and STIM1-deficient npTh17 and pTh17 cells. P (adj.) < 0.10.

(B) Volcano plots of DEGs in npTh17 cells (gray dots). Highlighted in red (upregulated in Stim1fl/flCd4Cre) and blue (downregulated in Stim1fl/flCd4Cre) are DEGs that belong to a gene expression signature of npTh17 cells and pTh17 cells defined previously (Gaublomme et al., 2015, Lee et al., 2012).

(C) Normalized expression of Th17 cell-associated cytokines and receptors in npTh17 cells derived from WT and Stim1fl/flCd4Cre mice. Bar graphs represent the mean ± SEM from 3 mice per group. Statistical analysis by unpaired Student's t-test with the following significance levels: *p<0.05, ***p<0.001.

(D) Summary of KEGG pathway analysis of npTh17 and pTh17 cells. Venn diagram shows pathways that are deregulated in one or both Th17 subsets. P (adj.) < 0.05 for all pathways. Some unique and shared pathways are indicated.

(E) KEGG network analysis of npTh17 cells. P (adj.) < 0.05 for all pathways.

(F) Heat map of DEGs (p (adj.) < 0.1) within the KEGG pathways "Foxo signaling" and "HIF-1 signaling" that are specifically deregulated in npTh17 cells. Numerical values are relative gene expression.

(G) Gene set enrichment analysis (GSEA) of DEGs in WT and STIM1-deficient npTh17 cells. Normalized enrichment score (NES) and p-values are as indicated for the following genesets: Myc targets: "Menssen_myc_targets"; Glycolysis: "Humancyc_mm_glycolysis_i"; Mitochondrial function: "Wong_mitochondria_gene_module"; TCA cycle: "Kegg_mm_citrate_cycle".

Data information: Statistical analysis in (A,F) by Wald t-test and correction using the Benjamini-Hochberg method; in (D,E) by hypergeometric distribution and correction using the Benjamini-Hochberg method; in (G) by phenotype-based permutation test.
STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of Th17 cells (2020)
Figure 8

Figure 8. STIM1 controls aerobic glycolysis and oxidative phosphorylation in non-pathogenic murine Th17 cells and human T cells.

(A) Heat map of metabolism-associated DEGs (p (adj.) < 0.10) in npTh17 cells of WT and Stim1fl/flCd4Cre mice determined by RNA-Seq as described in Figure 7. Heatmap shows relative minimum and maximum values per gene (row min/max). Numerical values are relative gene expression. TCA, tricarboxylic acid.

(B-D) Glucose and mitochondrial metabolism in npTh17 cells from WT (grey or black) and Stim1fl/flCd4Cre (blue)mice differentiated for 3 days in vitro. (B) Glucose uptake by npTh17 cells were loaded with 2-NBDG (+) or not (-) and analyzed by flow cytometry 90 min later. Bar graphs show the delta MFI of 2-NBDG fluorescence normalized to unlabeled cells. Data represent the mean ± SEM from 3 independent experiments. (C) Extracellular acidification rate (ECAR) of npTh17 cells before and after addition of 25 mM glucose, 5 μM oligomycin and 100 mM 2-deoxy-D-glucose (2-DG). Seahorse graphs and bar graphs depicting glycolysis, glycolytic capacity and reserve the mean ± SEM from of 2-3 mice per genotype from one representative out of three independent experiments. (D) Oxygen consumption rate (OCR) of npTh17 cells before and after addition of 1 μM oligomycin, 1.5 μM FCCP and 100 nM rotenone / 1 μM antimycin. Seahorse graphs and bar graphs depicting basal and maximal respiration, coupling efficiency and ATP production represent the mean ± SEM from of 2-3 mice per genotype from one representative out of three independent experiments.

(E-H) Glucose and mitochondrial metabolism in human CD4+ T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 24 hours before analysis. Some HD T cells were stimulated in the presence of 1μM FK506. (E,F) ECAR before and after addition of 25 mM glucose, 5 μM oligomycin and 100 mM 2-DG. Seahorse graphs (E) and bar graphs (F) represent the mean ± SEM of 3 (HD), 4 (P1) and 2 (HD+FK506) technical replicates from isolated T cells from one experiment. (G,H) OCR before and after addition of 1 μM oligomycin, 1.5 μM FCCP and 100 nM rotenone / 1 μM antimycin. Seahorse graphs (G) and bar graphs (H) depicting basal and maximal respiration, SRC and ATP production (right) represent the mean ± SEM of 2 technical replicates from isolated T cells from one experiment.

Data information: Statistical analysis in (A) by Wald t-test and correction using the Benjamini-Hochberg method; in (B-D) by unpaired Student's t-test with the following significance levels: *p<0.05, **p<0.01, ***p<0.001.

Expanded View Figure Legends
Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility (2017)
Figure 1

Figure 1.

Microtubule plus-end tracking of dynein in the presence of dynactin and EB protein

(A) Time series of TIRF microscopy images and (B) individual and dual colour kymographs showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio). (C) Merged triple colour and single fluorescence channel kymographs showing microtubule end tracking of GFP-dynein (green in merge) and Alexa647-EB3 (red in merge) on dynamic Alexa568-microtubules (blue in merge). Concentrations were 10 nM GFP-dynein, 10 nM human dynactin, 10 nM Alexa647-EB3, and 17.5 µM tubulin. (D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of either dynactin (D) or EB proteins (E). Concentrations of the proteins present as in A. (F) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of both human dynactin and EB1 (blue squares, as in A), in the absence of dynactin (red circles, as in D), or in the absence of an EB protein (black triangles, as in E). Mean values from at least two separate experiments per condition (with a total of at least 30 kymographs) are shown; error bars are s. e. (G) Kymographs of GFP-dynein microtubule end tracking in the absence of ATP (in contrast to all other experiments that contain ATP). Other conditions as in A. For merged kymographs, microtubule plus and minus ends are labelled by (+) and (−). Experiments were performed at 30°C.
Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility (2017)
Figure 2

Figure 2.

Effect of BicD2-N on microtubule end tracking of dynein in the presence of dynactin and EB1

(A) Merged triple colour and single fluorescence channel TIRF microscopy kymographs of GFP-dynein showing end tracking, processive motility and diffusive and static binding in the presence of all DDB components and EB1. Protein concentrations are 10 nM GFP-dynein (green in merge), 20 nM human dynactin, 20 nM EB1, 200 nM Alexa647-BicD2-N (magenta in merge), and 17.5 µM Alexa568-tubulin (blue in merge). Alexa647-BicD2-N often co-localises with processively moving and occasionally with statically bound GFP-dynein, but not with plus end-tracking GFP-dynein. (B) Averaged fluorescence intensity profiles of GFP-dynein (green squares) and Alexa647-BicD2-N (magenta circles) at growing microtubule plus ends. Mean values from three separate experiments per condition (with a total of at least 75 kymographs) are shown; error bars are s. e. (C, D) Dual and single colour kymographs of an Atto565-microtubule (magenta) in the presence of GFP-dynein (green), human dynactin and EB1, and either (C) in the absence or (D) presence of 5 µM BicD2-N; other conditions as in A. The increased BicD2-N concentration in D compared to A strongly reduces microtubule plus end tracking of GFP-dynein. (E) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the condition without BicD2-N (red circles, as in C) or with 5 µM BicD2-N (black squares, as in D). Mean values from three separate experiments per condition (with a total of at least 106 kymographs) are shown; error bars are s. e. Microtubule plus and minus ends in merged kymographs are labelled by (+) and (−). Experiments were performed at 30°C.
Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility (2017)
Figure 3

Figure 3.

Spatial initiation probability of processive DDB runs

(A) Example dual colour kymograph and a schematic showing how the distance of initiation of processive runs was measured. The protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 5 µM BicD2-N, 20 nM EB1 and 17.5 µM Atto647-tubulin. (B) Histograms of the spatial initiation probabilities of processive runs within the first 7.2 µm from growing microtubule plus ends in the absence (black bars), or presence of 10 nM EB1 (red bars). 199 and 221 initiation positions were determined for the two conditions (from three experiments each). (C) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the conditions as in B. Mean values from three separate experiments per condition (with a total of at least 69 kymographs) are shown; error bars are s. e. Experiments were performed at 30°C. (D) Histogram of spatial initiation probabilities of DDB on static GMPCPP-microtubules. 294 initiation positions were detemined (from three experiments). Concentrations of DDB components as in B.
Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility (2017)
Figure 4

Figure 4.

p150-dependent plus end tracking of dynein

(A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (A) 10 nM GFP-dynein (green) tracking the growing end of a Atto565-microtubule (magenta) in the presence of pig dynactin at an elevated concentration of 80 nM. (B) 10 nM GFP-dynein (green) tracking the plus end of an Atto565-microtubule (blue) in the presence of 10 nM Alexa647-p150 (a fragment containing the first 517 amino acids of p150; called p150-N in the text) (red). Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods). (C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (C) In standard assay buffer, 10 nM Alexa647-p150 (green) accumulates at the plus end and on the GMPCPP-stabilised segment of a growing Atto565-microtubule (magenta). (D) At higher ionic strength (BRB80 supplemented with 60 mM KCl, see Methods), Alexa647-p150 (green) binds only weakly to the microtubule without a detectable plus end preference. Protein concentrations as in C. The Atto565-tubulin concentration was always 17.5 µM tubulin. The temperature was 30°C.
Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility (2017)
Figure 5

Figure 5.

Regulation of microtubule end tracking versus processive motility of dynein by Lis1 and BicD2-N in the presence of dynactin and EB1

(A-C) Kymographs showing GFP-dynein (green) on dynamic Atto647N-microtubules (magenta) in the presence of all the regulators at different Lis1 concentrations. Protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 20 nM EB1, 5 µM BicD2-N, and (A) either no Lis1, (B) 1 μM mCherry-Lis1, or (C) 5 μM mCherry-Lis1. Together with BicD2, Lis1 increases the number of processive dynein runs and high concentrations of Lis1 restore plus-end localisation of dynein in the presence of BicD2-N. Microtubule orientation as indicated. (D) Averaged intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of EB1, dynactin and 5 µM BicD2-N (black squares, as in A), in the presence of 1 µM mCherry-Lis1 (blue circles, as in B) and 5 µM mCherry-Lis1 (red triangles, as in C). Mean values from three separate experiments per condition (with a total of at least 49 kymographs) are shown; error bars are s. e. (E) The average number of processive DDB runs per µm microtubule length for the three different Lis1 conditions (as in A, B and, C). Error bars are s. d. *p = 0.001, **p = 0.003, ***p = 0.001 (unpaired t-test). 218, 969, and 646 processive DDB events were analysed for the three conditions, respectively (from two experiments each). (F) Bar graphs showing the mean velocity and the mean run length of processive DDB events for the three Lis1 conditions (as in A, B and C). Error bars are the s.e.m. Mean velocities were 0.37 ± 0.02 µm/s (A), 0.38 ± 0.03 µm/s (B), and 0.35 ± 0.03 µm/s (C). Mean run lengths were 3.1 ± 0.4 µm (A), 3.2 ± 0.5 µm (B), and 2.9 ± 0.3 µm (C). For each condition over 300 complexes were analysed from three different data sets. (G) Histogram of spatial initiation probabilities of DDB runs in the presence of EB1 and Lis1. 381 initiation positions were determined (from two experiments). Experiments were performed at 30°C.
Molecular determinants of selective clearance of protein inclusions by autophagy (2012)
f1

(a–d) Formation of protein inclusions in SY5Y cells expressing FL (a) or the indicated truncated forms of Sph1 (b–d) was induced by 12 h lactacystin treatment. After extensive washes, cells were maintained in serum-supplemented (serum+) or serum-free (serum−) media, and in the absence (Control) or presence of 10 mM 3-MA, an inhibitor of macroautophagy. Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (left) or Agg (right) observed under different recovery conditions. Values are mean+s.e. (n=4). *Significant versus serum+ condition. See also Supplementary Figs S1–S5.
Molecular determinants of selective clearance of protein inclusions by autophagy (2012)
f2

(a–c) Top: fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL (a), or the indicated truncated Sph1 proteins (b–c) treated with lactacystin (12 h) and recovered in basal conditions (serum+). Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (middle) or Agg (bottom). Values are mean+s.e. (n=3). *Significant versus control. (d–f) Relative fold difference in the number of FL (d) or truncated (e–f) Sph1 Agm (left) and Agg (right) in WT and Atg5 KO mice MEFs incubated in serum+ or serum− conditions. Values are mean+s.e. (n=3). *Significant versus serum+ condition.
Molecular determinants of selective clearance of protein inclusions by autophagy (2012)
f3

(a–d) Left: fluorescence images of SY5Y cells co-expressing HA-p38 (red) and GFP (green) forms of FL (a) or the indicated truncated Sph1 (b–d). Cells were treated with lactacystin (12 h) and recovered in the presence or absence of serum. Graphs: percentage of p38 existing alone in Agm or co-aggregating with Sph1 in the forms of Agm or Agg (left), and susceptibility of p38 existing on its own in Agm or co-aggregating with Sph1 proteins in Agm toward serum-stimulated autophagic clearance (right). Values are expressed as folds Agm in the presence of serum and are mean+s.e. (n=3). *Significant versus serum+ condition calculated using Student’s t-test. (e) Autophagic susceptibility of Agm formed by p38 and ANK1-p38 fusion proteins. Values are mean+s.e. (n=3). *Significant versus control group calculated using Bonferroni test. See also Supplementary Fig. S6.
Molecular determinants of selective clearance of protein inclusions by autophagy (2012)
f4

(a) Top: colocalization of LAMP1 (red) with Agm and Agg of ACA and CACT Sph1 proteins highlighted by anti-GFP (green) in SY5Y cells. Arrows indicate colocalization. Bottom: percentage LAMP1 colocalization with Agm and Agg in cells maintained in serum+ or serum− media, treated or not with lysosomal inhibitors (N/L). (b) Percentage LAMP1 colocalization with Agm in cells co-expressing HA-p38 and the indicated Sph1 proteins maintained in the absence of serum. (c) Left: colocalization of LAMP1 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Right: percentage LAMP1 colocalization with p38 and ANK1-p38 Agm in cells maintained in the presence or absence of serum. (d,e) Correlative light and electron microscopy of SY5Y cells expressing the indicated GFP-tagged Sph1 truncations (d), or p38 and ANK1-p38 (e). Top: phase-contrast fluorescence microscopy; middle: electron micrographs of the boxed region; bottom: higher magnification examples of autophagic vacuoles (yellow arrows). #Location of the Agm or Agg structures in both the fluorescence images and the corresponding micrographs. (f) Top: colocalization of LC3 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Bottom: percentage colocalization of LC3 with the fluorescent proteins. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,c,f) or p38 Agm (b) calculated using Bonferroni test. See also Supplementary Fig. S7.
Molecular determinants of selective clearance of protein inclusions by autophagy (2012)
f5

(a) Fluorescence images showing colocalization patterns of the indicated GFP-p38 variants and Sph1 (green) with endogenous p62 (red) in SY5Y cells. Arrows indicate colocalization. (b) Quantitative FRAP analysis of mCherry-p62 expressed alone (control) or co-expressed with the indicated GFP-Sph1 and -p38 variants. Top: time course of changes in relative fluorescence intensity (RFI); bottom: percentage of p62 in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Representative images are shown in Supplementary Fig. S8. (c–d) Top: IBs for p62 and actin in control and p62 knockdown (p62 KD #325 or #788) SY5Y cells (c); and in control and p62 KO MEFs (d). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells in response to serum removal. (e) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1. Arrows indicate colocalization. (f,g) Top: IBs for NBR1, p62 and actin in control and NBR1 knockdown (NBR1 KD #161) SY5Y cells (f), and in control and p62/NBR1 double knockdown (Double KD #A or #B) SY5Y cells (g). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells. For all densitometric analyses, values are mean+s.e. (n=3). *Significant versus serum+ condition. See also Supplementary Fig. S8–S11.