EMBO/soda-nel huggingface dataset:
SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity (2021)
Figure 5

Figure 5. SRPK1 inhibitors selectively suppress RAN translation.

(A) Schematic of SRPK1 signaling pathway that regulates subcellular SRSF1 localization, which in turns can impact export and translation of repeat RNAs in the cytoplasm. SRPK1 may also act directly on protein translation pathways (dotted arrow). Known pharmacological compounds that inhibit SRPK1 can disrupt this pathway.

(B) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3 biological replicates). Schematics of the AUG-nLUC-3xFLAG and +1CGG(100)-nLuc-3xFLAG reporters presented on top.

(C) Relative expression of AUG-nLuc and CGG-nLuc reporters in HEK293T cells (n=8-9 biological replicates) following treatment with DMSO and SRPIN340.

(D) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top.

(E) Immunoblot of DMSO and SPHINX31 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3 biological replicates).

(F) Anti-FLAG of DMSO and SPHINX31 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates).

Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in sample buffer.
SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity (2021)
Figure 6

Figure 6. SRPK1 inhibition prevents stress-induced enhancement of RAN translation.

(A) Expression of +1CGG-nLuc-3xFLAG RAN translation reporters in HEK293T cells treated with 2 μM TG (for stress induction) analyzed by immunoblot (n=3 biological replicates). To evaluate effects of SRPK1 inhibition cells were pre-treated with DMSO or SRPIN340 before reporter transfection.

(B) Relative expression of +1CGG-nLuc reporters in HEK293T cells (n=6 biological replicates) following stress induction with 2 μM TG treatment. Values normalized to vehicle (DMSO) treatment. As in (A), cells were pre-treated with DMSO or SRPIN340 before reporter transfection.

(C, D) Expression of GGGGCC-nLuc-3xFLAG (C) and +2CGG-nLuc-3xFLAG (D) RAN translation reporters in HEK293T cells treated with 2 μM TG (for stress induction) analyzed by immunoblot (n=3 biological replicates). To evaluate effects of SRPK1 inhibition cells were pre-treated with DMSO or SRPIN340 before reporter transfection.

(E) SRPK1 inhibition by SRPIN340 did not alter eIF2α phosphorylation in response to 2 μM TG (for stress induction) in HEK293T cells by immunoblot (n=3 biological replicates).

(F) SPRK1 inhibition suppressed eIF2α phosphorylation in HEK293T cells treated with 2 μM TG (for stress induction) in the presence of the +1CGG reporter. Cells were pre-treated with DMSO or SRPIN340 before reporter transfection (n=3 biological replicates).

Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.
SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity (2021)
Figure 7

Figure 7. SRPK1 inhibition alters CGG repeat RNA localization and directly inhibits RAN translation.

(A) Immunoblot of cytoplasmic phospho-SRSF1/2 after SRPIN340 treatment. GAPDH is used as the loading control. Error bars represent mean +/- SD (n=3 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05

Full images of the same blot showing levels of all phospho SR proteins (pan SRSF phosphorylation) after SRPIN340 treatment are presented in Figure EV 4A.

(B) ICC images of FLAG-tagged SRSF1 after SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1 signal (see methods for details). Error bars indicate mean +/- 95% CI (n= 85-126 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.

(C) Nucleocytoplasmic distribution CGG repeat RNA after SRPIN340 treatment compared to vehicle (DMSO) as detected by HCR. Quantification shows the ratio of nuclear and cytoplasmic intensity of CGG RNA signal (see methods for details) as parts of whole. Error bars indicate mean +/- 95% CI (n= 124-151 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.

(D) Anti-FLAG immunoblot blot of DMSO and SRPIN340 pre-treated HEK293T cells transfected with in vitro transcribed CGG-nLuc-3xFLAG reporter RNA. β-Actin is used as a loading control. Error bars represent mean +/- SD (n=6 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05

(E) Relative expression of in vitro transcribed CGG-nLuc reporter in HEK293T cells pre-treated with DMSO and SRPIN340. Error bars represent mean +/- SD (n=9 biological replicates).

(F) Expression of in vitro transcribed Aug-nLuc and CGG-nLuc reporters in rabbit reticulocyte lysate (RRL) in vitro translation system following pre-treatment with DMSO or SRPIN340. Error bars represent mean +/- SD (n=9 biological replicates).

Data information: For E and F, Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01
SRSF protein kinase 1 modulates RAN translation and suppresses CGG repeat toxicity (2021)
Figure 8

Figure 8. SRPK1 inhibition mitigates (CGG)100 RNA toxicity in primary rat cortical neurons.

(A, B) Pharmacological targeting of SPRK1 with (A) 40 μM SRPIN340 or (B) 8 μM SPHINX31 reduced the cumulative risk of death in +1(CGG)100-EGFP (encoding FMRpolyG) expressing neurons. n= # of neurons quantified for each condition; Cox proportional hazard analysis, ∗∗∗p < 0.001.
Mapping the origin and fate of myeloid cells in compartments of the eye by single-cell profiling (2020)
Figure 3

Figure 3: Prenatal source of eye macrophages.

Left: Creation of Acta1GFP/+:Acta1+/+ bone marrow chimeras. Right: Donor-derived GFP+CD45loCD11b+ were detectable in the recipient retina by flow cytometry 20 weeks after bone marrow reconstitution. FACS Plots are representative for five animals from one experiment.

Quantification of GFP+ cells among CD45+CD11b+ retinal microglia by flow cytometry. Data are presented as mean ± s.e.m. One symbol represents one mouse.

Typical retinal flat mount from Acta1GFP/+:Acta1+/+ bone marrow chimeras 20 weeks after reconstitution. Donor-derived GFP+Iba1+ cells (arrow) and GFP-Iba1+ resident microglia (asterisks) are shown. Pictures are representative for three animals from one experiment. Scale bars represents 50 µm.

Microscopy-based quantification of GFP+Iba1+ retinal microglia in the inner (IPL) and outer plexiform layer (OPL). One symbol represents one mouse. Data are presented as mean ± s.e.m.

Scheme of a fate mapping experiment using Cx3cr1CreERT2:Rosa26-YFP female mice. tamoxifen (TAM) and progesterone injection were performed at embryonic day 9.0 (E9.0). Mice were subsequently evaluated at postnatal day 0 (P0). Administration of TAM leads to intra-embryonic excision of a stop sequence flanked by loxP sites (grey triangles) in Cx3cr1 expressing cells which causes stable and steady YFP expression under the control of the Rosa26 promotor.

Direct fluorescence microscopic visualization for YFP (green), the macrophage marker Iba1 (red) and DAPI for the nuclei (blue) at P0. YFP+Iba1+ double-positive cells are marked by arrows. YFP-Iba1+ single-positive cells are labelled by asterisks. Representative images out of five examined animals are shown. Scale bars represents 25 µm.

Quantitative analysis of regional YFP expression in Iba1+ macrophages in TAM-induced and untreated Cx3cr1CreERT2:Rosa26-YFP mice. Bars represent means ± s.e.m. Quantification was done from three (untreated) or five (TAM) mice obtained from one (untreated) or two (TAM) independent experiments. Level of significance determined by Mann-Whitney test between TAM and untreated revealed *p < 0.05 and Kruskal-Wallis test between retina, ciliary body and cornea revealed *p = 0.0204.
Mapping the origin and fate of myeloid cells in compartments of the eye by single-cell profiling (2020)
Figure 9

Figure 9: Correlative analysis between murine experimental and human neovascular CNV.

a) H2-Aa expression visualized by trajectory analysis and represented in a t-SNE plot.

b) H2-Aa immunohistochemistry (green) by Iba1+ (red) microglia inside lesioned and non-lesioned areas in the retina and RPE. Arrows indicate microglia with aquired MHCII expression whereas asterisks mark microglial cells that remained MHCII negative. Picture is representative of two mice. In the RPE, arrow heads label Tom-MHCII+ cells. Scale bars represents 25 µm.

c) HLA-DRA mRNA expression examined by RNA-Seq via Massive analysis of cDNA ends (MACE) analysis of human formalin-fixed and paraffin-embedded (FFPE) membranes of choroidal neovascularization (CNV). Bars represent means ± s.e.m. of four investigated human samples of age-related macular degeneration associated with choroidal neovascularization and four control tissues consisting of choroid and RPE. Level of significance was calculated using DESeq (p = 3.61x10-21).

d) Immunohistochemistry for HLA-DR in the RPE/choroid of one control and the CNV membrane of one patient suffering from age-related macular degeneration associated with choroidal neovascularization.

EXPANDED VIEW FIGURE LEGENDS
Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex (2009)
figf1

(a) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1–EGFP in Becn1−/−;Becn1–EGFP/+ mice, as detected by an anti-Beclin 1 antibody. (b) Coomassie-stained SDS–PAGE showing the Beclin 1-interacting proteins immuno-isolated from brain and liver of the 'rescued' mice (lanes 2 and 4) and of control Becn1+/− littermates (lanes 1 and 3), using an anti-GFP antibody. Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15/p150 (band 1), Vps34/class III PI(3)K (band 3), UVRAG (band 4), Beclin 1–EGFP (band 5), Atg14L (band 6, asterisk, gi|27369860) and Rubicon (band 2, asterisk, gi|45708948). UVRAG levels varied with different affinity-purification conditions, suggesting an unstable association of UVRAG with the complex. (c) Schematic representations of the domain structures of Atg14L and Rubicon. Atg14L contains two coiled-coil domains (CCD1 and CCD2), which are also homologous with the SMC domain (structural maintenance of chromosomes). Rubicon contains an N-terminal RUN (for RPIP8, UNC-14 and NESCA) domain, a C-terminal Cys-rich domain and a central CCD domain. (d) Western blot analysis of Atg14L, Rubicon, Vps34 and Beclin 1 in gel filtration fractions from wild-type mouse liver extract showed co-elution of these proteins in fractions 38–45. Atg14L was also eluted in later fractions 51–56. The fractions for the peak elution of thyroglobulin (670K) and γ-globulin (158K) are indicated by arrows. Control siRNA-transfected NIH 3T3 cell lysate was loaded as a positive control (labelled as 1) for the migration position of the Atg14L protein on SDS–PAGE; Atg14L siRNA-transfected NIH 3T3 cell lysate was loaded as a negative control (labelled as 2). (e, f) Co-immunoprecipitation confirmed protein–protein interaction between Atg14L and Rubicon. HEK 293 cells were co-transfected with Atg14L–EGFP and Flag–Rubicon (e) or Rubicon–EGFP and Flag–Atg14L (f). Cell lysates were used for immunoprecipitation with an anti-GFP antibody and the resulting immunoprecipitates were blotted with an anti-Flag antibody. Our results show immunoprecipitation of Rubicon by Atg14L (e) and vice versa (f). WCL, whole cell lysate; IP, immunoprecipitated. See Supplementary Information, Fig. S6 for full scans of blots in a, d, e and f.
Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex (2009)
figf2

(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under normal and nutrient-starvation conditions in NIH 3T3 cells. (b) Compared with control siRNA, Atg14L siRNA decreased long-lived protein degradation in NIH 3T3 cells under normal (*P = 0.007) and starvation (*P = 5 × 10−6) conditions (one-tailed Student's t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA, 10 mM), a PI(3)K inhibitor. (c) Vps34 kinase assay. HEK 293T cells were co-transfected with Myc–Vps34–Vps15 and Flag–Atg14L or Flag vector, either in the absence or in the presence of Beclin 1–EGFP. Myc–Vps34–Vps15 was immunoprecipitated by anti-Myc antibody for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by thin-layer chromatography (TLC), quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34 as measured by western blot (upper panel). The quantified results (lower panel) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2.5-fold, but only when Beclin 1 was also overexpressed (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 5). (d) Colocalization of co-expressed Atg14L–EGFP (green) and Beclin 1–AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar, 10 μm. (e) Electron microscopy images show large structures (asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L–EGFP and Beclin 1–AsRed: concentric membrane 'rings' (panel 1); two large structures (3–5 μm in diameter, panel 2) containing material with high electron density (inset, enwrapping double membranes); numerous autophagosomes (arrows, panel 3) in the cytoplasm; immuno-electron microscopy image of a Atg14L–Beclin 1 structure (labelled with anti-GFP antibody and developed by DAB, panel 4) enwrapped with concentric membrane 'rings'. M, mitochondria; N, nucleus. Scale bar, 500 nm. (f, g) EGFP–Atg12 (f) or EGFP–Atg5 (g) (green) was colocalized with the large structures (arrows) that were labelled by Atg14L–AsRed (red) and Beclin 1–Myc (blue) in transfected HeLa cells. Some of these structures seemed to be 'ring'-shaped (yellow arrows and inset). Scale bar, 10 μm. See Supplementary Information, Fig. S6 for full scans of blots in a and c.
Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex (2009)
figf3

(a) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient-starvation conditions. (b) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient-starved conditions in HEK 293 cells either stably expressing (upper rows) or transiently transfected with (lower rows) Rubicon–EGFP. The control cells were either stably expressing or transiently transfected with the EGFP–N3 vector. (c) Vps34 kinase activity. HEK 293T cells were co-transfected with Myc–Vps34–Vps15 and Flag–Rubicon or Flag vector, either in the absence or in the presence of Beclin 1–EGFP. Myc–Vps34–Vps15 was immunoprecipitated by an anti-Myc antibody and used for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by TLC, quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34, as measured by western blotting (upper panel). The quantified results (lower panel) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0.58-fold, but only without Beclin 1 overexpression (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 4). (d) Effect of overexpressing Flag–Rubicon on autophagosome acidification, as monitored by mCherry–GFP–LC3 fluorescence. HeLa cells were transiently co-transfected with mCherry–GFP–LC3 and Flag–Rubicon (or control Flag vector). Cells co-expressing mCherry–GFP–LC3 and control Flag vector contained many red-only puncta along with yellow (indicating the presence of both red and green) puncta, suggesting the presence of both autolysosomes and nascent autophagosomes (upper panel). In contrast, cells co-expressing mCherry–GFP–LC3 and Flag–Rubicon contained primarily yellow or white puncta, suggesting the presence of only nascent autophagosomes (lower panel, white arrows). Notably, some cells, which were co-transfected with mCherry–GFP–LC3 and Flag–Rubicon but expressed high levels of mCherry–GFP–LC3 and undetectable levels of FLAG–Rubicon, contained many red-only puncta (lower panel, yellow arrows). (e) Quantification of the results in d show that overexpressing Flag–Rubicon markedly reduced the percentage of red-only puncta (mCherry–LC3) from 39% in the control Flag vector-transfected cells to 2% in the Flag–Rubicon-transfected cells (*P = 2 × 10−26, one-tailed Student's t-test with unequal variances, n = 30), indicating that overexpression of Rubicon blocks autophagosome acidification or maturation. See Supplementary Information, Fig. S6 for full scans of blots in a–c.
Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex (2009)
figf4

(a) Colocalization of Rubicon–EGFP-associated structures with the late endosome/lysosome marker Lamp1 (arrows) in HeLa cells transfected with Rubicon–EGFP. Note that some of the Rubicon–EGFP-associated structures show a 'ring' shape (yellow arrows). Scale bar, 10 μm. (b) Partial colocalization of Rubicon–EGFP-associated structures with the MVB marker LBPA (arrows) in HeLa cells transfected with Rubicon–EGFP. Scale bar, 10 μm. (c) Representative ultrastructural images show aberrant expansion of late endosomal/lysosomal structures in HEK 293T cells overexpressing Rubicon–EGFP. These abnormal organelles are large in size, with high (orange arrows) or low (black arrows) electron density. Some enclose small vesicles (purple arrows) and some resemble the MVB (blue arrows). Scale bars, 500 nm. (d) Representative ultrastructural images show late endosome/lysosome-like structures that are labelled with anti-GFP gold particles (panels 3, 4) in HEK 293T cells transiently transfected with Rubicon–EGFP. These structures are enwrapped by double membranes (panel 4 inset) and co-labelled by anti-GFP (developed by DAB) and anti-Lamp1 (gold enhanced) (panels 5–7) antibodies. Note that mitochondria are mostly negative for Rubicon–EGFP (panel 4). The negative controls are without antibody (panels 1–2). M, mitochondria. Scale bars, 200 nm.
Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex (2009)
figf5

(a) Local sequence alignment between the C-terminal Cys-rich domain of Rubicon and FYVE domains of several known FYVE-containing proteins. Rubicon does not possess the key consensus sequences of a typical FYVE domain, that is, N-terminal WxxD, central R[R/K]HHCR and C-terminal RVC (indicated by red bars). (b) Colocalization of the PI(3)P-enriched lipid domain marker p40 (phox)-PX–EGFP (green) and Rubicon–AsRed (red) on large punctate structures (arrows) in the co-transfected HeLa cells (upper panels). Treatment with the PI(3)K inhibitor wortmannin (75 nM) for 1 h caused disappearance of the PI(3)P-enriched lipid domains, whereas the Rubicon–AsRed-positive structures were maintained (lower panels). Scale bars, 10 μm. (c) Subcellular localization of transiently transfected Rubicon–EGFP, RubiconΔRUN–EGFP, RubiconΔC–EGFP or RubiconΔRUNΔC–EGFP in HeLa cells. In contrast to punctate Rubicon–EGFP and RubiconΔRUN–EGFP, RubiconΔC–EGFP and RubiconΔRUNΔC–EGFP were dispersed in the cytoplasm. ΔRUN, RUN domain deletion; ΔC, cysteine-rich domain deletion. Scale bars, 10 μm. (d, e) Absence of full-length Beclin 1 (d) or Beclin 1-CE mutant (e) (red) on the Rubicon–EGFP-positive structures (green) in HEK 293 cells stably expressing Rubicon–EGFP. These cells were transiently transfected with either Beclin 1–AsRed (d) or Flag–Beclin 1-CE (e; that is, the Flag-tagged Beclin 1 mutant containing both CCD and ECD, which mediate the Beclin 1–Rubicon interaction as shown in Supplementary Information, Fig. S3h). Scale bars, 10 μm. (f) The formation of the Rubicon–EGFP-positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 cells stably expressing Rubicon–EGFP. (g) A model for the Beclin 1–Vps34 protein complexes and their functions. Note that this model is not intended to propose a direct binary interaction. In this model, a core Beclin 1 complex is composed of Vps34/PI(3)K, p150/Vps15, Beclin 1, UVRAG and probably substoichiometric Atg14L (indicated by the tight binding and functional connection between Atg14L and Beclin 1). Under physiological conditions, a large Beclin 1–Vps34 complex is formed, including the core complex and Rubicon. This large complex may be reduced to form smaller complexes, such as an Atg14L–Beclin 1-containing complex and a Rubicon-containing complex. These smaller complexes may be the functional units participating in autophagy regulation through modulating the Vps34 lipid kinase activity. See Supplementary Information, Fig. S6 for full scans of blots in f.
Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism. (2021)
Figure 1

Figure 1. Tyr-Asp inhibition of GAPC activity is associated with the shift of the glycolytic flux towards the PPP and increased NADPH/NADP+ ratio.

(A) GAPC enzymatic activity was measured in wild-type crude extracts treated with H2O (mock), and 100 µM of Tyr-Asp, Tyr, Asp, and Ile-Glu.

(B-D) The enzymatic activities of GAPC (B), GAPA/B (C), and GAPN (D) were measured in the wild type and double k.o. mutant gapc1gapc2 treated with H2O (mock), and 100 µM of Tyr-Asp.

(E) Schematic representation of glycolysis and pentose phosphate pathways.

(F) Glucose-labeled C6 and C1 flux experiment in 10-day-old Arabidopsis seedlings. C6/C1 ratio for control (water treated) and Tyr-Asp (100 µM) treated wild type seedlings after 35, 70 and 145 min is shown.

(G) NADPH/NADP+ ratio.

(H) NADH/NAD+ ratio. Ratios were calculated from the NAD+, NADH, NADP+, and NADPH measurements.

Data information: Data are mean ± SEM of n=4 (A-D; four technical replicates), n=3 (F; three independent flasks), and n=5-6 (G-H; five to six independent seedlings flasks). For (A-D, F), significance was assessed using unpaired two-tailed Student t-test. For (G-H), significance was assessed using two-way ANOVA (p≤0.05; letters show significance associated with the Tyr-Asp treatment). G6P: glucose 6-phosphate; F6P: fructose 6-phosphate; FBP: fructose 1,6-bisphosphate; DHAP: dihydroxyacetone-phosphate; G3P: glyceraldehyde 3-phosphate; 1,3bisPGA: 1,3bis-phosphoglycerate; 3PGA: 3-phosphoglycerate; PPP: pentose phosphate pathway; R5P: ribose 5-phospate. In G and H, C stands for control; n.s.: not significant.