EMBO/soda-nel huggingface dataset:
The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization (2021)
Figure 3

Figure 3. GAR domain interactions with phospholipid membranes.

A. Molecular dynamics simulations of native (R4) or variant (K4 and N4) nucleolin GAR-derived peptides interacting with an idealized phospholipid bilayer (posphatidylcholine (PC): phosphatidylserine (PS), 4:1) in water. Amino acid sequences of peptides are shown above. Shaded area - standard deviations of three independent runs, each 50 ns long. Mean values are indicated by colored lines and overlay of these is shown on the right.

B. Flow cytometry histograms of TAMRA-labeled peptide uptake into HEK-293 cells after 1h incubation at 4°C in growth medium.

C. Quantification of mean fluorescence intensities in (B). n = 6 independent biological repeats; means ± SEM; * p< 0.05, *** p< 0.001, **** p< 0.0001, ANOVA with Tukey's post-test.

D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm.

E. Quantification of (D). n = 3 independent biological repeats; means ± SEM; * p<0.05, paired Student's t-test a. u. - arbitrary units.

F. Analysis of cell surface nucleolin in HEK-293 cells. Cells were treated with 0.5 mM sulfo-NHS-SS-biotin for 30 min at 4°C, lysed and subjected to affinity pulldown of biotinylated proteins with streptavidin Dynabeads, eluted by 50 mM DTT and probed in Western blot with antibodies indicated on the left. Endogenous nucleolin (Ncl) was readily detected in the biotinylated membrane protein pool. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and importin β1 (Impβ1) served as negative controls.

G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl FL), HA-Dendra2-nucleolin with a GAR domain deletion (Ncl ΔGAR) or HA-Dendra2-nucleolin with all 10 arginines in the GAR domain mutated to asparagines (Ncl GAR(N)) and processed as in (F).

H. Quantification of (G). Levels of nucleolin in pulldowns were normalized to input levels and expressed as % relative to full-length HA-Dendra2-nucleolin. n = 4 independent biological repeats; means ± SEM; **** p<0.0001, ANOVA with Dunnett's post-test.

See also Fig. EV2 and Appendix Fig. S3.
The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization (2021)
Figure 4

Figure 4. GAR domain and nucleolar localization of nucleolin.

A. GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons, transduced with the peripheral neuron specific AAV-PHP.S vector expressing full length HA-Dendra2-nucleolin (Ncl FL) or respective ΔGAR mutant (Ncl ΔGAR) upon seeding and analyzed by epifluorescence microscopy 9-10 days later. Shown are representative Dendra2 fluorescence images collected from the non-activated (green) emission line. Hoechst 33342 (10 µM) was used to outline nuclei. Superimposed phase contrast and Dendra2 fluorescence images are shown on the right. Scale bar - 25 µm.

B. Quantification of the nucleolar/nuclear intensity ratios in DRG cells shown in (A). n = 38-54 cells per sample in 3 independent biological repeats; means ± SEM; **** p<0.0001, Mann-Whitney test.

C. Time-lapse live imaging of nuclei in DRG neurons transduced with AAV-PHP.S HA-Dendra2-nucleolin (full length and ΔGAR mutant), after a hypotonic challenge with double-distilled water (ddH20). Shown are epifluorescence images (Dendra2 green emission line) taken at indicated times after medium replacement with ddH20. Scale bar - 10 µm.

D. Quantification of nucleolar Dendra2 signal in (C) with intensity values immediately before medium replacement set as 100%. n = 17-24 cells per sample in 3 biological repeats; means ± SEM; Two-way ANOVA with Sidák's post-test detected a significant time-dependent reduction of nucleolar signal (p < 0.0001) and no difference between full length and ΔGAR mutant nucleolin (p > 0.05).

See also Appendix Fig. A4.
The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization (2021)
Figure 5

Figure 5. Reduced levels of axonal nucleolin in nucleolin GAR+/- mice.

A. Targeted deletion of nucleolin GAR domain by CRISPR-Cas9. Schematic shows mouse nucleolin exons targeted by single guide RNAs (sgRNAs) and resulting deletion in the GAR domain amino acid sequence.

B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size).

C. Quantification of (B), total nucleolin levels (sum of both bands where applicable) were normalized to Tuj1 and expressed as GAR+/- / WT ratios. n = 3 independent biological repeats; means ± SEM; * p < 0.05, 1 sample t-test.

See also Appendix Fig. S5.
The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization (2021)
Figure 6

Figure 6. Reduced levels of nucleolin mRNA cargos in axons of nucleolin GAR+/- mice.

A, B. Representative maximum projections of exposure matched confocal images of immunofluorescence for nucleolin and neurofilament (NF) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR+/- mice shown in (A). Upper panels show total nucleolin stain. Middle panels show merged image of nucleolin (gray), NF and Tuj1 (magenta), and DAPI (blue). Lower panels show nucleolin overlaps with NF and Tuj1 signals as the "axon only" signal. B shows quantification of nucleolin immunofluorescence with approximately a 50% reduction in nucleolin in the axons of GAR+/- mice in vivo. n = 3 WT, n = 4 GAR+/-; means ± SEM; * p < 0.05, unpaired Student's t-test. Scale bar - 10 µm.

C-F. Representative, exposure matched maximum projection confocal images of FISH for Kpnb1 (C) or mTOR (E) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT (left) or GAR+/- mice. Upper panels for each show total mRNA signal. Middle panels show mRNA (gray) signals merged with NF plus Tuj1 (magenta) and DAPI (blue). Lower panels show mRNA signal that overlap with NF plus Tuj1 signal (labeled "axon only" signal). Quantification of axonal Kpnb1 (D) and mTOR (F) mRNA signals compared to the negative control, DapB mRNA, show a significant reduction in these axonal mRNAs in the GAR+/- mice. n = 3 WT, n = 3 GAR+/-; means ± SEM; ** p < 0.01, *** p < 0.001, ****p < 0.0001 unpaired Student's t-test. Scale bar - 10 µm.

See also Appendix Fig. S6.
The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization (2021)
Figure 7

Figure 7. Axonal transcripts associated with the Ncl-Kif5a complex.

A. Workflow for profiling mRNAs bound by the Ncl-Kif5a complex. Nucleolin (Ncl) and Kif5a-binding RNA was isolated from wild type (WT) adult mouse sciatic nerve axoplasm by immunoprecipitation; in addition, DRG neurons from adult WT and GAR+/- mice were cultured in modified Boyden chambers and RNA was isolated from cell body and axonal sides. RNA-seq analysis from the resulting four datasets yielded 11,771 overlapping transcripts. The latter were further processed into a subset of 488 transcripts enriched in Ncl and Kif5a pulldown and depleted in axons of nucleolin GAR+/- mice compared to WT (B). For a detailed workflow please see Fig. EV3.

B. Clustering of 488 Ncl/Kif5a-enriched transcripts reduced in GAR+/- versus WT axons. Yellow cluster - transcripts not significantly enriched in the soma of GAR+/- DRG neurons versus the WT control, Purple cluster - transcripts enriched in the soma of GAR+/- DRG neurons versus the WT control. Heatmap shows mean log2 fold changes across four datasets (see also Fig. EV3A), from left to right: 1) nucleolin-binding mRNAs in mouse sciatic nerve axoplasm; 2) Kif5a-binding mRNAs in mouse sciatic nerve axoplasm; 3) mRNA abundance in DRG neuron cell bodies of GAR+/- mice relative to abundance in WT mice; 4) mRNA abundance in DRG neuron axons of GAR+/- mice relative to abundance in wild type mice. All transcripts chosen for this cluster analysis showed significant enrichment in Ncl IP and reduction in GAR+/- axons versus WT, as determined by Rank-Rank Hypergeometric Overlap (RRHO) as well as a fold-change in Kif5a IP versus control > 2.

C. Genes comprising the purple cluster - transcripts significantly enriched by both Ncl and Kif5a immunoprecipitation and showing a reduction in GAR+/- axons concurrent with an enrichment in GAR+/- soma (compared to the WT control). Inpp5f (highlighted in red) was chosen for follow-up.

D. Representative images for FISH analysis of Inpp5f in DRG neurons treated for 48h with 10 µM AS1411 or 10 µM control aptamer, replated and grown for 18h. FISH signal is shown in gray, cell somata (left) and axons (right) are visualized by neurofilament immunostaining (magenta). Scr - scrambled FISH probe served as a negative control. Scale bar - 10 µm.

E. Quantification of axonal Inpp5f signal in (D) as density of RNA granules along axons. n = 11-19 cells per sample; means ± SEM; * p<0.05, unpaired Student's t-test.

F. RT-qPCR analysis of axonal Inpp5f mRNA levels in DRG neurons grown in Boyden chambers and treated for 48h with 10 µM AS1411 or 10 µM control aptamer. Axon/cell body ratios of Inpp5f normalized to Gapdh levels are shown. n = 4 independent biological repeats; means ± SEM; * p<0.05, unpaired Student's t-test.

See also Fig. EV3.
The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization (2021)
Figure 8

Figure 8. Wild-type, but not ΔGAR mutant nucleolin rescues defective axonal localization of its cargo mRNAs in DRG neurons treated with nucleolin siRNA.

A, B. Representative exposure-matched FISH/IF images for DRG neurons co-transfected with control vs. nucleolin (Ncl) siRNA plus siRNA-resistant wild type (WT; A) or ∆GAR (B) Ncl cDNAs. Axonal Kpnb1 mRNA is decreased with the Ncl knockdown, and this is not rescued by expression of ∆GAR Ncl mutant (scale bars - 10 µm).

C, D, Quantification of RNA signals from FISH/IF for Kpnb1, mTOR, and Inpp5f in cell bodies (C) and axons (D) for DRG cultures transfected as in A are shown. Axons of neurons cotransfected with Ncl siRNA plus ∆GAR show significantly lower signals for each mRNA compared to Ncl siRNA plus WT and control siRNA plus WT or ∆GAR; N ≥ 10 for cell bodies, N ≥ 25 for axons over 3 biological repeats; means ± SEM; **** p ≤ 0.001 by one-way ANOVA with Tukey HSD post hoc.

See also Figs EV4 and EV5, and Appendix Fig. S7
The Glycine Arginine Rich Domain of the RNA Binding Protein Nucleolin Regulates its Subcellular Localization (2021)
Figure 9

Figure 9. Reduced levels of full length axonal nucleolin increase axonal outgrowth in DRG neurons.

A. Cultured DRG neurons from wild type (WT) Thy1-YFP mice and Thy1-YFP / GAR+/- mice (YFP signal is shown in green). Cells were imaged every hour for a period of 48 h. Scale bar - 100 μm.

B. Quantification of the time-lapse imaging experiment shown in (A). Growth rate of the longest neurite of each cell was calculated from the time point of starting growth. n = 4 independent biological repeats; means ± SEM, **** p < 0.0001, two-way ANOVA.

C. Fluorescence images of cultured DRG neurons from adult C57BL/6 mice infected with AAV-PHP.s expressing HA-Dendra fused with the wild type nucleolin GAR domain (GAR WT) or with the GAR domain with all 10 arginines substituted with asparagines, GAR(N). Neurons were re-plated 9-10 days after AAV infection. 24h after re-plating cells were fixed and stained with anti-HA (grey) and anti-NFH (magenta) antibodies. Scale bar - 100 μm.

D. Quantification of total axonal outgrowth and mean process length in HA-positive cells in (C). Outgrowth measurements were based on NFH staining. n = 6 independent biological repeats; means ± SEM; * p < 0.05, *** p < 0.001 (Student's-test).
PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability (2021)
Figure 1

Figure 1. Lipid metabolism pathways are impaired in PKM2 deficient cells.

(A) Heatmap showing the altered metabolic pathways in PKM2 KO MCF7 and MDA-MB-231 cells relative to the parental cells (n=4). Fatty acid metabolism (Acyl Carnitine) and long chain fatty acid pathways were significantly changed in both cell lines.

(B) Heatmap showing the differential fatty acid β-oxidation pathway metabolite levels between PKM2 KO MCF7 and MDA-MB- 231 cells, and their respective parental counterparts (n=4).

(C) Decreased levels of free carnitine and modified carnitine species in PKM2 KO cell lines as compared with their parental counterparts (n=4).

(D) Heatmap showing the decreased levels of long chain fatty acids in PKM2 KO cell lines relative to their parental counterparts (n=4).

(E) Increased acetyl-CoA in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=4).

(F) Decreased total cholesterol in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=3).

(G) Confocal imaging of neutral lipid droplets stained with BODIPY493/503 or Nile red dye in PKM2 KO cells relative to the parental cells. Data represent one of three independent experiments with similar results. Scale bar, 25μm.

Data information: Data are displayed (C, E and F) as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.

Source data are available online for this figure.
PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability (2021)
Figure 2

Figure 2. SREBPs activation is impaired in PKM2 KO breast cancer cells.

(A) Heatmap showing the differential gene expression patterns between MDA-MB-231 PKM2 KO and parental cells (n=3).

(B) Gene set enrichment analysis of the top enriched gene signatures associated with PKM2 knockout in (A). Hallmark 50 gene sets and KEGG 186 gene sets were used for this analysis.

(C) Decreased cholesterol homeostasis signature in MDA-MB-231 PKM2 KO cells. Cholesterol homeostasis signature was generated by GSEA (v4.1.0) with adjusted p-value and normalized enrichment score (NES).

(D) Heatmap showing the differentially expressed genes related to cholesterol homeostasis in PKM2 KO vs. parental MDA-MB-231 cells (n=3).

(E) Volcano plot showing proteomic changes in PKM2 KO vs. parental MDA-MB-231 cells. Blue triangle symbol indicated PKM2 level; TMEM33, PKM1 and RNF5 were labeled in red dots. Horizonal dashed line indicated p-value 0.05 and vertical dashed lines indicated 2-fold cutoff. (n=3).

(F) Correlation of PKM2 deficiency induced protein changes between MCF7 and MDA-MB-231 cells. Green highlights down-regulated lipid-related proteins. PKM1, TMEM33, OCIAD2 and TIMM8A were up-regulated and highlighted in red dots.

(G) Western blot analysis of TMEM33, ABCA1, SREBP1, HMGCR, SCAP, FASN, LDLR, FDFT1, I DI1, RNF5, INSIG 1 and 2, and Calnexin in PKM2 KO MCF7 and MDA-MB-231 cells, and their respective parental cells. Arrow indicates nuclear SREBP1. P: precursor, N: nuclear.

Source data are available online for this figure.
PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability (2021)
Figure 3

Figure 3. TMEM33 is the downstream effector of PKM2 controlling lipid metabolism gene expression.

(A) Immunofluorescence of SREBP1 in MCF7 parental and PKM2 KO cells by confocal imaging. Scale bar, 5μm.

(B) Quantitation of nuclear SREBP1 intensity in A. n>=25 cells as in (A) from three different fields.

(C) Western blot analyses of nuclear and cytoplasmic SREBP1 levels in parental and two PKM2 KO clones. PCNA serves as a nuclear protein loading control.

(D) Western blot analyses of TMEM33, RNF5, SCAP, SREBP1, and IDI1 in PKM2 KO MCF7 and MDA-MB-231 cells stably transfected with shControl (shCtrl) or shTMEM33 constructs. Arrow indicates nuclear SREBP1, N: nucleus and P: precursor.

(E) Cell proliferation assays of the PKM2 KO MCF7 cells stably transfected with shCtrl or shTMEM33 constructs (n=3). shCtrl: shControl.

(F) Cell migration assays of PKM2 KO MDA-MB-231 cells stably transfected with shCtrl or shTMEM33 constructs (n=3). Scale bars, 50µm.

(G) Western blot analysis of TMEM33, RNF5, SCAP, SREBP1, HMGCR, LDLR, FDFT1, IDI1, and PKM2 in wild type, PKM2 KO, TMEM33 OE (pool), and two TMEM33OE clones (clones #1 and #8) MDA-MB-231 cells. Arrow indicates nuclear SREBP1. N: nucleus and P: precursor.

(H, I) Gene set enrichment analysis of the top enriched gene signatures (H), including (I) Cholesterol homeostasis signature in MDA-MB-231 TMEM33 OE (#1) cells using KEGG and hallmark gene sets in GSEA databases. Adjusted p-value and normalized enrichment score (NES) were generated by GSEA.

(J) Volcano plot showing the altered transcripts induced by TMEM33 OE (clone #1) (p value <0.05) and PKM2 KO (p value <0.05) in MDA-MB-231 cells. Green depicts shared down- regulated genes by both TMEM33 OE and PKM2 KO; red depicts shared up-regulated genes by both TMEM33 OE and PKM2 KO. Variance analysis (ANOVA) was used to calculate the p-value.

(K) Volcano plot showing protein changes induced by TMEM33 OE (p value <0.05) and PKM2 KO (p value <0.05) in MDA-MB-231 cells. TMEM33 labeled in red dot was upregulated in both TMEM33 OE and PKM2 KO cells and PKM2 labeled in purple had no change in TMEM33 OE cells. ANOVA was used to calculate the p-value.

Data information: Data are displayed (B and E) as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001.

Source data are available online for this figure.
PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability (2021)
Figure 4

Figure 4. TMEM33 recruits RNF5 to promote SCAP polyubiquitination.

(A) Increased SCAP polyubiquitination in TMEM33 transfected HEK293T cells. 293T cells were transiently transfected with Halo-tagged TMEM33 for 18 hours followed by treatment with MG-132 for 3 hours.

(B) Western blot analysis of PKM2, PKM1, NRF1, TMEM33, RNF5 and SCAP levels in MEF (PKM2fl/fl, Cre-ERT2) cells treated with 4-OHT in a time course experiment.

(C) Co-immunoprecipitation of endogenous SCAP and RNF5 in Flag-tagged TMEM33 stably expressed MCF7 cells. SEC61β is an ER protein, which is found to interact with TMEM33.

(D) Reciprocal co-immunoprecipitation of TMEM33 and RNF5 in MDA-MB-231 cells. TMEM33, RNF5 and SCAP were detected by Western blotting.

(E) Proximity ligation assays of TMEM33 and RNF5 in MDA-MB-231 cells using confocal imaging. NC means negative control. Scale bar, 10μm.

(F) Interaction between in vitro translated TMEM33 and RNF5. FLAG-tagged vector or TMEM33 and HA-tagged RNF5 were expressed using an in vitro transcriptional and translational kit, followed by FLAG antibody pull down.

(G) Increased polyubiquitination of SCAP in RNF5-transfected HEK293 cells. 293T cells were transiently transfected HA-tagged RNF5 for 18 hours followed by MG-132 treatment for 3 hours. Arrow indicated HA-tagged RNF5.

(H) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T cells. 293T cells were stably transfected with shCtrl or shRNF5 constructs, then the indicated plasmids were co-transfected into shCtrl or shRNF5 expressing 293T cells for 18 hours followed by MG-132 treatment for 8 hours. FLAG M2 beads were used for pull-down.

(I) The relative binding affinity of FLAG-tagged full-length and truncated SCAP proteins with endogenous TMEM33 and RNF5. The band intensity of TMEM33 and RNF5 in Appendix Fig S2D were quantified and normalized with the FLAG control. SCAP protein encompasses eight transmembrane (TM) helices in N-terminus (TM 2-6: sterol-sensing domain) and a WD40 domain in C-terminus with total of 1279 aa.

(J) The relative binding affinity of HA-tagged full- length and truncated TMEM33 with endogenous SCAP and RNF5. The band intensity of SCAP and RNF5 in Appendix Fig S2E were quantified and normalized to the HA control. TMEM33 protein encompasses three transmembrane (TM) helices in N-terminus and a C-terminal cytosolic domain with a total of 247 aa.

Source data are available online for this figure.
PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability (2021)
Figure 5

Figure 5. RNF5 promotes SCAP degradation in breast cancer cells.

(A) Lysine 48 linked polyubiquitinated SCAP levels in parental and PKM2 KO MDA-MB-231 cells.

(B) SCAP-GFP stability in parental and PKM2 KO MDA-MB-231 cells (n=5). Cells stably expressing SCAP-GFP were treated with cycloheximide (CHX). GFP signal was measured at the indicated time.

(C) Western blot analyses of RNF5 and SCAP in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells.

(D) qRT-PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells (n=3).

(E) Western blot analyses of RNF5, SCAP and IDI1 in Ctrl and HA-RNF5 OE MCF7 cells.

(F) qRT-PCR analyses of SREBPs downstream genes in Ctrl and HA-RNF5 OE MCF7 cells (n=3).

(G) Cell proliferation of Ctrl and HA-RNF5 OE MCF7 cells measured by MTT assays (n=8).

Data information: Data are displayed (B, D, F and G) as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Source data are available online for this figure.
PKM2-TMEM33 axis regulates lipid homeostasis in cancer cells by controlling SCAP stability (2021)
Figure 6

Figure 6. PKM2/VCP/RNF1 axis regulated TMEM33 expression.

(A) Western blot analyses of NRF1, TMEM33 and PKM2. The two bands of NRF1 denote precursor and cleaved NRF1.

(B) The relative mRNA expression levels of TMEM33, RNF5, MT1A and MT2A in parental and PKM2 KO MDA-MB-231 cells (n=3).

(C) Regulation of TMEM33 promoter with or without an ARE motif by NRF1 in luciferase assays in 293T cells (n=3).

(D) Recruitment of NRF1 to TMEM33 promoter analyzed by ChIP-qPCR in parental and PKM2 KO MCF7 cells. IgG was used as a negative control. NRF1 enrichment on the TMEM33 promoter region was normalized to IgG control (n=3).

(E) Western blot analyses of TMEM33 and RNF5 in NRF1 overexpressed MCF7 clones.

(F) Western blot analyses of TMEM33 and RNF5 in shCtrl and shNRF1 PKM2 KO MCF7 cells.

(G) Co-immunoprecipitation of VCP with FLAG-tagged PKM1/2 in PKM2 KO 293T cells. FLAG-tagged PKM1 or PKM2 construct was transiently transfected into PKM2 KO or parental 293T cells for 24 hours, followed by pull-down using FLAG-M2 beads. PKM1, PKM2 and VCP antibodies were used for immunoblotting.

(H) Decreased VCP and PKM2 in vitro interaction by treatment with TEPP-46.

(I) TEPP-46 treatment attenuates accumulation of polyubiquitinated proteins by NMS-873 detected with anti-Ub western blot. MCF7 cells were treated with DMSO, NMS873 (5μM), TEPP-46 (10μM), or NMS873 (5μM) plus TEPP-46 (10μM) for 7 hours followed by co-treatment with cycloheximide (CHX, 50 μg/ ml) for one hour.

(J) Western blotting of VCP, NRF1, TMEM33 and PKM2 in MCF7 treated with TEPP-46 for 24 hours.

(K) A schematic diagram depicting the regulation of lipogenesis by PKM2. ER located PKM2 inhibits VCP activity, which is essential for NRF1 precursor cleavage. Cleaved NRF1 acts as a transcriptional factor for TMEM33. ER localized TMEM33 inhibits SREBPs activity through promoting SCAP degradation in RNF5-dependent manner. Knocking out PKM2 leads to increased NRF1 cleavage and TMEM33 expression. Consequentially, SCAP is destabilized by RNF5-mediated ubiquitination and expression levels of lipid synthetic genes are decreased.

Data information: Data are displayed (B-D) as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Source data are available online for this figure.