EMBO/soda-nel huggingface dataset:
Regulators of autophagosome formation in Drosophila muscles. (2015)
Fig 7

Glycogen metabolism enzymes are autophagy regulators in skeletal muscle.

(A) Primary cultured muscles with control lacZ RNAi, treated for 12 hrs with Rap+CQ. Note the formation of large GFP-Atg8a vesicles throughout the muscles. (B) GFP-Atg8a vesicles are reduced in primary cultured muscles with GlyS RNAi, treated for 12 hrs with Rap+CQ. Knockdown of Pfk (C) or CG9485 (D) causes increased accumulation of GFP-Atg8a vesicles in primary cultured muscles treated for 12 hrs with Rap+CQ. (E) Quantification for A-D of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). SEM is indicated with *p<.05; **p<.01. (F-H)
Dmef2-Gal4 drives expression of UAS-GFP-Atg8a in animals starved on low nutrient food + 1 mg/ml CQ for 6 hrs. (F) Control white RNAi muscles accumulate large autophagosomes at 1 mg/ml CQ. (G) RNAi knockdown of CG9485 increases autophagosome accumulation in the muscle. (H) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in the muscle. (I) Quantification of the GFP-Atg8a punctae area in muscles from white, CG9485, and AGBE knockdowns.n = 6 larval muscles with SEM indicated and *p<.05. In all panels green = GFP-Atg8a and red = Phalloidin. (J) Western blot analysis of endogenous Atg8a from third instar muscle lysate. Dmef2-Gal4 driven knockdown of AGBE but not CG9485 inhibits Atg8a lipidation, indicating reduced autophagy levels.
Binding of the Atg1/ULK1 kinase to the ubiquitin‐like protein Atg8 regulates autophagy (2012)
Figure 2

Atg1 binds Atg8 in a LIR‐dependent manner. (A) Schematic representation of budding yeast Atg1 and sequence alignment of its putative LIR motif with Atg1 homologues from various species. Conserved residues of the predicted LIR motif are indicated with grey boxes, residues targeted by mutagenesis are marked with asterisks (*). The Atg1 kinase domain is underlined and the Atg13‐binding region is shaded in black. Circled ‘P’ mark known phosphorylation sites on Atg1. N: amino‐terminus; C: carboxy‐terminus. (B) GST‐Atg8 or GST‐Ubiquitin (Ub) was purified from E. coli and bound to GSH beads. Wild‐type Atg1‐TAP or the VE and EYE mutants were purified from yeast, incubated with the immobilized GST proteins and bound proteins analysed by western blotting. (C) GST‐Atg8 and GST‐Ubiquitin were immobilized as described in (B), and probed for their ability to bind Atg1‐TAP isolated from wild‐type or atg13Δ cells. (D) atg1Δatg13Δ cells expressing as indicated wild‐type Atg1‐TAP or the VE or EYE mutants, and wild‐type Atg13 or the Atg13‐FV mutant were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 was determined by western blotting. (E) GST, GST‐LC3B, GST‐GATE16 and GST‐GABARAP were purified from E. coli and bound to beads. HEK293 cell lysates prepared from cells expressing wild‐type (wt) HA‐ULK1 or the DFP mutant were incubated with the beads, and bound proteins analysed by western blotting. (F) HEK293 cells were co‐transfected with GFP or GFP‐GABARAP along with either wild‐type HA‐ULK1 or the DFP mutant. GFP and GFP‐GABARAP were immobilized on GFP‐Trap resin and HA‐ULK1 binding was analysed by western blotting. Figure source data can be found with the Supplementary data.
Binding of the Atg1/ULK1 kinase to the ubiquitin‐like protein Atg8 regulates autophagy (2012)
Figure 3

The LIR domain in Atg1 is functionally important in vivo. (A) atg1Δatg13Δ cells containing an empty control plasmid, wild‐type or the indicated Atg1 mutants, and Atg13 wild‐type, the Atg13‐FV mutant or an empty plasmid were grown to mid log phase. Processing of endogenous Ape1 was analysed by western blotting, and quantified as described in the legend of Figure 1I. Expression of the different proteins was controlled by immunoblotting with antibodies against Atg1 or Atg13. The asterisk marks a non‐specific band detected by the Atg13 antibody. (B) pho8Δ60pho13Δatg1Δ cells expressing wild‐type (wt), kinase‐dead (kd), Atg1‐VE (VE) or ‐EYE (EYE) mutants or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in ‘Materials and methods’, and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls. (C) Cells as in (A) expressing GFP‐Atg8 were grown to mid log phase with starvation for 4 h in SD‐N medium. The processing of GFP‐Atg8 was analysed and quantified as in Figure 1I. (D) Exponentially growing atg1Δ and atg1Δatg8Δ strains expressing the PAS marker Ape1‐RFP and either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were examined by fluorescence microscopy. Bar=5 μm. (E) atg1Δ cells containing TAP‐tagged wild‐type or the indicated Atg1‐mutants were grown to mid log phase. Atg1 was immunoprecipitated and its in vitro autophosphorylation activity was measured by autoradiography. The kinase‐dead (kd) Atg1‐mutant K54A serves as a negative control. Figure source data can be found with the Supplementary data.
Binding of the Atg1/ULK1 kinase to the ubiquitin‐like protein Atg8 regulates autophagy (2012)
Figure 4

Atg1/ULK1 localize to autophagosomes in yeast and mammals. (A) Exponentially growing ypt7Δ cells were starved in SD‐N medium for 4 h, lysed and the extract separated into a cytoplasmic (S) and a 5000 g membrane pellet fraction. The pellet was treated with (+) or without (−) TX‐100, centrifuged and the supernatant (S) and pellet (P) fractions analysed by western blotting with anti‐Atg1, anti‐Pep12 and anti‐Pgk1 antibodies. (B) Exponentially growing ypt7Δatg1Δ cells expressing wild‐type Atg1 or the Atg1‐VE mutant protein were starved in SD‐N for 4 h, lysed and the extract separated into cytoplasmic (S) and membrane (P) fractions. The fractions were analysed by western blotting with anti‐Atg1, anti‐Pgk1 and anti‐Ape1 antibodies, and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild‐type cells. (C) HEK293 cells were co‐transfected with GFP‐GABARAP and HA‐ULK1, starved for 2 h, and subsequently immunostained for HA (red) and WIPI2 (white). The arrows mark putative autophagosomes that stain positive for all three markers. Bar=10 μm. (D) HEK293 cells stably expressing GFP‐LC3 were transfected with wild‐type HA‐ULK1 or the DFP mutant, and immunostained for HA and WIPI2 after 2 h starvation. HA‐ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells (from one out of two experiments) and plotted as the ratio of ULK1 to WIPI2 puncta. ***Indicates a statistically significant P‐value of 0.0001. One of the two independent experiments is shown. (E) Quantification of HA‐ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild‐type HA‐ULK1 or the DFP mutant. Cells were treated as above, and the spot number counted using Imaris software. A representative immunofluorescence image is shown in Supplementary Figure S3F. *** and * indicate P‐values of 0.0001 or 0.0158, respectively. Figure source data can be found with the Supplementary data.
Binding of the Atg1/ULK1 kinase to the ubiquitin‐like protein Atg8 regulates autophagy (2012)
Figure 5

Atg1 travels with autophagosomes to the vacuole during starvation. (A) atg1Δ cells containing either wild‐type Atg1 or the kinase‐dead Atg1‐T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD‐N. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting. Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux. (B) atg1Δ cells containing either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown and analysed as in (A). (C) atg1Δpep4Δ cells containing YFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown to log phase and starved in SD‐N. Cells were monitored by time‐lapse microscopy and the vacuolar accumulation of Atg1 was quantified as described in ‘Materials and methods’. Data are plotted as the mean±s.e.m. from at least 90 quantified cells. Additional images are shown in Supplementary Figure S3H. Bar=5 μm. (D) atg1Δatg13Δ cells expressing GFP‐tagged wild‐type Atg13, and either wild‐type Atg1 or the Atg1‐VE mutant (left panel) and wild‐type cells containing Atg13‐GFP and either wild‐type Atg13 or Atg13‐FV were grown to mid log phase followed by starvation for 6 h in SD‐N media. GFP cleavage was analysed by western blotting. Figure source data can be found with the Supplementary data.
RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex (2021)
Figure 1

Figure 1. Rgcc expression during the embryonic mice and human cortical development

A, Expression pattern of Rgcc in the embryonic day 14.5 (E14.5) mouse cortex as detected by in situ hybridization. The right panels showed the expression patterns of NSC markers, including Pax6, Sox2, and Ki67, along the ventricular zone (VZ) of the E14.5 mouse cortex. Scale bar: 20 μm.

B, Analyzing single cell transcriptomic database of the E14.5 mouse cortex, showed the specific expression of Rgcc in NSCs. Data were downloaded form http://zylkalab.org/datamousecortex.

C, RGCC expression and distribution in developing human fetal neocortex. RGCC microarray data were downloaded from BrainSpan Atlas of the Developing Human Brain (Available from: human.brain-map.org; Donor id: 14751, donor name H376.IIIB.02, donor age 16pcw). Two independent probes NO: A_23_P204937 and NO: A_23_P10137 were used. Heatmap was further prepared by MeV. Only the expressions along neocortex were shown.

D, Single cell RNA-seq analysis of human fetus cortex (https://cells.ucsc.edu/?ds=cortex-dev). Cell-type clusters expressing RGCC was circled by red dash line. Expression patterns of NSC markers were showed in the right panels.

E, Immunofluorescent staining of hCOs of day30. RGCC and PAX6 were co-stained. Scale bars: 100 μm.

F, Immunofluorescent staining of hCOs of day30. RGCC and TBR1 were co-stained. Scale bars: 250 μm.

Data information: Abbreviation: VZ, ventricular zone; GE, ganglionic eminence.
RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex (2021)
Figure 2

Figure 2. Knocking down Rgcc suppresses NSC proliferation and promotes NSC proneuronal differentiation

A, Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. Ki67 were co-stained with GFP. To quantify the ratio between NSC markers and GFP positive cells, only the cells in VZ and SVZ are taken into account. And ROIs of 800*1900 pixel were randomly picked for counting. Scale bars: 20 μm.

B, Quantification of the relative ratio of Ki67 and GFP double positive cells versus GFP positive cells.

C, Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Sox2 were co-stained with GFP. Scale bars: 20 μm.

D, Quantification of the relative ratio of Sox2 and GFP double positive cells versus GFP positive cells.

E, Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Pax6 were co-stained with GFP. Scale bars: 20 μm.

F, Quantification of the relative ratio of Pax6 and GFP double positive cells versus GFP positive cells.

G, Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 5 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E12.5. All the brains were harvest at E17.5. Tbr1 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Tbr1 positive region for counting. Scale bars: 100 μm.

H, Quantification of the relative ratio of Tbr1 and GFP double positive cells versus GFP positive cells.

I, Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Ctip2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Ctip2 positive region for counting. Scale bars: 100 μm.

J, Quantification of the relative ratio of Ctip2 and GFP double positive cells versus GFP positive cells.

K, Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Satb2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along ventricle for counting. Scale bars: 100 μm.

L, Quantification of the relative ratio of Satb2 and GFP double positive cells versus GFP positive cells.

M, Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. Scale bars: 100 μm.

N, The ratio of GFP positive cells in VZ/SVZ, IZ, and CP were counted for migration analysis.

Data information: Data are presented as mean ± SEM; n≥3 mouse brains for each group; Statistical significance was tested by Student's t-test. **, p<0.01; ***, p<0.001. Abbreviation: VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; n.s., not significant.
RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex (2021)
Figure 6

Figure 6. The subcellular co-localization of RGCC protein

A, RGCC was co-stained with PAX6 on proliferating rosettes of hCOs at day 30. RGCC exhibits specifically higher expression level on NSCs around apical surface (marked by arrow head) adjust to ventricle-like region (marked by white dash line). Scale bar: 50 μm. Representative image of one M-phase NSC with higher RGCC expression around nucleus were show in right panel and marked by yellow arrow head. Scale bar: 5 μm.

B, RGCC was co-stained with PCNT on 3D hCO slices. RGCC was with stronger expression in NSCs during M-phase. prometaphase (B'), metaphase (B'') anaphase (B''') and telophase (B''''). RGCC was expressed around nucleus in interphase (B''''') . NSCs undergoing prometaphase, metaphase, anaphase and telophase were pointed out by yellow arrow. Interphase were pointed out by white arrow (B'''''). Immunostaining of 2D-differentiated NSCs in metaphase showed partial colocalization of RGCC with α-TUBULIN in the spindle (B'''''', yellow arrow). Scale bar: 5 μm.

C, The Western blots results. Proteins were collected from WT hCOs and RGCC KO hCOs #6 at day 45. B-ACTIN were used as references.

D, PCNT was co-stained with β -TUBULIN on WT NSCs and RGCC KO NSCs differentiated by 2D cultured dual-SMAD inhibition method. Scale bar: 5μm.

E, Representative images of measuring fluorencense intensity of β -TUBULIN and PCNT. Step-by-step details can be found in the Materials and Methods section. Scale bar: 5μm.

F, Quantification of fluorescence intensity of β -TUBULIN positive spindle fibers.

G, Quantification of mean fluorescence intensity of β -TUBULIN positive spindle.

H, I, Quantification of fluorescence intensity of PCNT positive centrosomes. Data were presented as mean ± SEM. Each plot represents the average intensity of two PCNT positive region in one dividing cell (n=11 for H9 cells; n=12 for RGCC-KO cells).Statistical significance was tested by one-way ANOVA. ****, p<0.0001.

Expanded View Figure legends
Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells. (2011)
Figure 8

Analysis of MiaPaCa-2 cell organelles after ultracentrifugation.

(A) Overlay of the proton NMR spectrum fragments of the following suspensions (from top to down): untreated first (lysosomal) cell fraction after ultracentrifgation and one washing (F1*), iodixanole (Optiprep) suspension, lower (F3), middle (F2) and upper fraction (F1) of the ultracentrifugates after two washings. The chemical shifts of the spectra were calibrated to the Choline/Phosphatidylcholine (Cho/PC) signal. Apart from iodixanol, we could identify the PUFA groups (methylene groups situated between two CH = CH groups), Cho/PC and glycerophosphocholine (GPC) in F1* and F1–3. Furthermore we observed lactate and the methylene groups of fatty acids (at 1.3 ppm), citrate (at 2.55 and 2.7 ppm) and phosphoethanolamine (at 3.1 ppm) in the F1* but not in the F1–F3 groups.. (B) Western blot analysis of LAMP-1, a late endosome marker, which was nearly identically distributed over the first two fractions compared to Cathepsin-D, in the controle group, but ocurred only in the first fraction in the OMP treated group. β-COP as an indicator of the Golgi-complex, is found strongly in the lower fraction in the control group, but very weakly in the OMP treated group. Cathepsin - D, an early endosome marker, which can be found in the upper and middle fraction of the control group, but regarding the OMP group, it was only found in the first one. (E) Overlay of NMR spectra of all fractions of control and OMP. The most relevant difference is the weaning of the GPC signal after OMP treatment in the 1st fraction after only 6 hours.
Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells. (2011)
Figure 9

Western Blot analysis with LC3B-antibody in ASPC-1 and MiaPaCa-2 cells.

The experiment was repeated three times with nearly identical results. The LC3-I fractions can be clearly distinguished above the LC3-II signals. Bafilomycin A1 elevated slightly the LC3-II signal intensity compared to control in both cell lines. There was a marked and dose-dependent increase of the signal strength of both fractions when OMP was used. 5-FU alone did not relevantly influence the LC3-level. The corresponding β-Actin level is shown below the LC3-WB to confirm the correctness of this semiquantitative evaluation.
Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells. (2011)
Figure 10

Gene expression analysis of membranal transport and apoptosis relevant genes in ASPC-1 cells.

Quantification of mRNAs in ASPC-1 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. The stars indicate significant differences after 24 hours compared to control. The pro-apoptotic bad-mRNA was upregulated after 5-FU- and 5-FU+OMP-treatment. The antiapoptotic bcl-2 mRNA was downregulated by 5-FU and bcl-XL by 5-FU+OMP. OMP led to downregulation of bad and survivin and to upregulation of the mdr-1 mRNA.
Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells. (2011)
Figure 11

Gene expression analysis of membranal transport and apoptosis relevant genes in MiaPaCa-2 cells.

mRNA quantification in MiaPaCa-2 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. While the mdr-1 mRNA is not signicantly changed, the vATPase mRNA is upregulated in the 5-FU+OMP group after 24 hours compared to control.
Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells. (2011)
Figure 12

Gene expression analysis of autophagy and apoptosis relevant genes by Western blot analysis.

The Bad, Puma and Atg12 proteins were detected in MiaPaCa-2 and ASPC-1 cells. The Bad expression is slightly increased by 5-FU and suppressed by OMP and 5-FU+OMP in ASPC-1, but not in MiaPaCa-2 cells. Puma is induced by OMP in both cell lines indicating involvement of the BH3-only pathway. Atg12 is strongly enhanced by OMP in both cell lines. Abbreviations: K - control, F - 5-FU, O - OMP, FO - 5-FU+OMP.
Engagement of TRAIL Triggers Degranulation and IFNγ Production in Human Natural Killer Cells (2022)
Figure 1

Figure 1 │ NK cell degranulation is associated with increased TRAIL surface expression after co-incubation with autologous HIV-1-infected CD4 T cells

Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry.

(A) Representative histograms (overlay) displaying CD107a expression on NK cells after culture with the described culture conditions (NK(-), Mock, HIV). For the HIV-1 co-culture condition, subsequent analyses of antigen expression were conducted by gating non-responsive (CD107a-) and responsive (CD107a+) NK cell subsets.

(B) Relative frequency of CD107a+ NK cells (y-axis) after culture in the described conditions (x-axis) (n = 19 different donors).

(C) HIV-specific response of bulk NK cells displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 19 different donors).

(D) Summary table showing numeric results of the 327 surface antigens analyzed. 48 surface molecules showed statistically significant intra-donor differences >5 p.p. in expression between CD107a+ and CD107a- NK cells after exposure to HIV-1-infected CD4 T cells (HIV).

(E) Representative histograms (overlay) showing TRAIL surface expression as fluorescence intensity on NK cells after co-incubation with HIV-1-infected CD4 T cells. Histograms show TRAIL expression for CD107a+ and CD107a- NK cell subsets (solid lines) as well as their corresponding FMO controls (dotted lines). Gate defining TRAIL+ cells is indicated as well.

(F) Left panel: Relative frequency of TRAIL+ cells (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells. Right panel: Difference in relative frequency of TRAIL+ cells (y-axis) between CD107a+ and CD107a- cells displayed as p.p. (n = 19 different donors).

(G) Left panel: TRAIL surface expression (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells displayed as median fluorescence intensity. Right panel: Differences in TRAIL surface expression (y-axis) between CD107a+ and CD107a- cells displayed as fold-difference (n = 19 different donors).

Data information: Wilcoxon signed-rank test. Adjustment for multiplicity: Benjamini and Hochberg false discovery rate (FDR) (D; F, left panel:); Bonferroni (F, right panel:; G). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.