Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet:

Article Snippet: His-Rac1(WT) , Cytoskeleton , Cat# RC01.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Activation Assay, BIA-KA, Mass Spectrometry, Plasmid Preparation, Software, Control, Transfection, Western Blot

Journal: bioRxiv

Article Title: PP2A B55δ Responsible for the High Initial Rates of Alcoholic Fermentation in Sake Yeast Strains of Saccharomyces cerevisiae

doi: 10.1101/402081

Figure Lengend Snippet: Effects of modification of the TORC1-Greatwall pathway on fermentation progression. Fermentation was monitored by measuring carbon dioxide emission. (A) Fermentation profiles of strain TM142 (wild type; gray) and its rim15 Δ disruptant (red). (B) Fermentation profiles of strain TM142 in YPD20 medium in the absence (wild type, gray) or presence (red) of 1 nM rapamycin. (C to J) Fermentation profiles of strain TM142 (wild type; gray) and its tor1 Δ (C), TOR1 L2134M (D), TOR2 L2138M (E), gtr1 Δ (F), gtr2 Δ (G), npr2 Δ (H), npr3 Δ (I), or sch9Δ (J) mutant (red). (K) Fermentation profiles of strain TM142 rim15 Δ in YPD20 medium in the absence ( rim15 Δ; gray) or presence (red) of 1 nM rapamycin. (L) Fermentation profiles of strain TM142 rim15 Δ ( rim15 Δ; gray) and its TOR1 L2134M mutant (red). (M, N) Fermentation profiles of strain IB1401 (wild type; gray) and its gtr1 Δ /gtr1 Δ(M) or sch9Δ/sch9Δ (N) disruptant (blue). (O, P) Fermentation profiles of the wild-type S. pombe strain (wild type; gray) and its tor2 E2221K (O) or sck1/2 Δ (P) mutant (green). Fermentation tests were performed in YPD20 medium (A to N) or in YPD10 medium (O, P) at 30°C for 5 d. Values represent the mean ± SD of data from two or more independent experiments. *, significantly different from the value for the control experiment ( t test, P < 0.05). Note that the experiments using laboratory, sake, and fission yeast strains are indicated in red, blue, and green, respectively. WT, wild type; Rap, rapamycin.

Article Snippet: Another S. cerevisiae laboratory strain X2180 and Schizosaccharomyces pombe wild-type strain 972 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Modification, Mutagenesis, Control

Journal: bioRxiv

Article Title: PP2A B55δ Responsible for the High Initial Rates of Alcoholic Fermentation in Sake Yeast Strains of Saccharomyces cerevisiae

doi: 10.1101/402081

Figure Lengend Snippet: Effects of modification of the Greatwall-PP2A B55Δ pathway on fermentation progression. Fermentation was monitored by measuring carbon dioxide emission. (A, B) Fermentation profiles of strain BY4741 (wild type; gray) and its rim15 Δ (A) or igo1/2 Δ (B) disruptant (red). (C, D) Fermentation profiles of the wild-type S. pombe strain (wild type; gray) and its cek1 Δ /ppk18 Δ (C) or igo1 Δ (D) disruptant (green). (E to I) Fermentation profiles of strain BY4741 (wild type; gray) and its pph21 Δ (E), pph22 Δ (F), tpd3 Δ (G), cdc55 Δ (H), or rts1 Δ (I) disruptant (red). (J to L) Fermentation profiles of the wild-type S. pombe strain (wild type; gray) and its ppa1 Δ (J), ppa2 Δ (K) or pab1 Δ (L) disruptant (green). (M, N) Fermentation profiles of strain BY4741 cdc55 Δ ( cdc55 Δ; gray) and its rim15 Δ (M) or igo1/2 Δ (N) disruptant (red). (O) Fermentation profiles of the S. pombe pab1 Δ strain ( pab1 Δ; gray) and its igo1 Δ disruptant (green). (P) Fermentation profiles of strain K701 UT-1T with an empty vector (wild type; gray) and with a functional RIM15 -expressing plasmid (blue). (Q, R) Fermentation profiles of strain K701 (wild type; gray) and its CDC55 WT /cdc55 MT δ (Q) or cdc55 WT δ /cdc55 MT (N) disruptant (blue). Fermentation tests were performed in YPD20 medium (A, B, E to I, M, N, P to R) or in YPD10 medium (C, D, J to L, O) at 30°C for 5 d. Values represent the mean ± SD of data from two or more independent experiments. *, significantly different from the value for the control experiment ( t test, P < 0.05). Note that the experiments using laboratory, sake, and fission yeast strains are indicated by red, blue, and green, respectively. WT, wild type.

Article Snippet: Another S. cerevisiae laboratory strain X2180 and Schizosaccharomyces pombe wild-type strain 972 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Modification, Plasmid Preparation, Functional Assay, Expressing, Control

Journal: bioRxiv

Article Title: PP2A B55δ Responsible for the High Initial Rates of Alcoholic Fermentation in Sake Yeast Strains of Saccharomyces cerevisiae

doi: 10.1101/402081

Figure Lengend Snippet: A hypothetical model of the regulation of fermentation control by the TORC1-Greatwall-PP2A B55δ pathway. Orange and green colors indicate higher and lower activities, respectively, than those of S. cerevisiae wild-type laboratory strains. (A) In S. cerevisiae laboratory strains and S. pombe , changes in the activity of TORC1, Greatwall, or PP2A B55δ may lead to altered alcoholic fermentation performance. (B) In S. cerevisiae sake strains, both the high TORC1 activity and the loss of Rim15p may contribute to the constitutively high PP2A B55δ activity. Thus, PP2A B55δ must be disrupted to impair the fermentation performance in these strains.

Article Snippet: Another S. cerevisiae laboratory strain X2180 and Schizosaccharomyces pombe wild-type strain 972 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Control, Activity Assay

Journal: bioRxiv

Article Title: Stabilization of AMPK/PFKL/RPIA in the Glycolytic Bodies Transduces IL6/STAT3 Signal in Hepatocarcinogenesis

doi: 10.1101/2024.02.29.582877

Figure Lengend Snippet: ( A ) Treatment with IL6 (20 ng/mL) for 2 hours (left panel) or the induction of cancer stem cells (CSCs) (right panel) upregulates the expression of most glycolysis-related genes, including PFKL, at the mRNA level. Red bars represent PLC5 cells after IL6 treatment or sphere formation, while black bars represent control cells. ( B ) IL6 treatment increases PFKL protein levels. The right panel shows the quantification results of western blot analysis for PFKL and RPIA protein levels. Black bars indicate no treatment control, red bars represent 0.5 hours of IL6 treatment, and blue bars represent 2 hours of IL6 treatment. ( C ) Knockdown of AMPK by shRNA reduces IL6-induced PFKL-upregulated protein expression in PLC5 cells, leading to decreases in pAMPK, PFKL, RPIA, pERK, and pSMAD5 levels, while it has no effect on STAT3 phosphorylation. The right panel illustrates the quantification of western blot results for AMPK, pAMPK, PFKL, RPIA, STAT3, pSTAT3, ERK, pERK, mTOR, p-mTOR, SMAD5, and pSMAD5. Black bars represent no treatment control, red bars indicate IL6 treatment, blue bars represent shAMPK, and the green bar denotes shAMPK+IL6. ( D ) Knockdown of AMPK decreases cell viability in PLC5 cells. Quantification of cell viability at 24, 48, and 72 hours is shown. Red bars represent shAMPK, while black bars denote shLuc control. ( E ) AMPK knockdown reduces IL6-stimulated cell viability, normalized to the control without IL6 treatment. Black bars represent the control without treatment, and red bars represent IL6 treatment. ( F ) Inhibition of AMPK with dorsomorphin decreases cell viability in three hepatoma cell lines. Cell death rates were quantified, and the IC50 for dorsomorphin in PLC5, Hep3B, and HepG2 cells is displayed in the upper left. ( G ) AMPK knockdown decreases migration ability with or without IL6 treatment. Quantification of migration without IL6 or with IL6 treatment is shown. Black bars represent sh-Luc control, and red bars denote sh-AMPK. ( H ) Suppression of AMPK with dorsomorphin reduces migration ability. Black bars represent the control without treatment, and red bars indicate dorsomorphin treatment. ( I ) Dorsomorphin significantly reduces PFKL protein levels. The left panel presents quantification of western blot results for AMPK, pAMPK, PFKL, SMAD5, pSMAD5, ACC, and pACC. Black bars represent the control without treatment, light red bars denote 1 µg/mL, and red bars indicate 6 µg/mL dorsomorphin treatment. ( J ) AMPK knockdown does not affect PFKL mRNA expression. Quantification of qPCR results for PFKL mRNA is shown. The red bar indicates dorsomorphin treatment, while the black bar denotes the control without treatment. ( K ) AMPK knockdown increases proteasome activity in PLC5 cells, suggesting that AMPK stabilizes PFKL by inhibiting proteasome activity. Quantification of proteasome activity is shown, with the red bar indicating shAMPK and the black bar representing shLuc control. Statistical analyses were performed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The specific antibodies against PFKL (Cell Signaling Technology Cat# 8175, RRID:AB_11178807), STAT3 (Cell Signaling Technology Cat# 9132, RRID:AB_331588), pSTAT3 (Cell Signaling Technology Cat# 9130, RRID:AB_330367), AMPK (Cell Signaling Technology Cat# 2603, RRID:AB_490795), pAMPK (Cell Signaling Technology Cat# 5759, RRID:AB_10949320), ERK (GeneTex Cat# GTX59618, RRID:AB_10726211), pERK (Abcam Cat# ab32538, RRID:AB_11156273), RPIA (Abcam Cat# ab67080, RRID:AB_1142656), PK (GeneTex Cat# GTX111536, RRID:AB_1951258), G6P (GeneTex Cat# GTX113203, RRID:AB_2037119), TPI (GeneTex Cat# GTX104618, RRID:AB_1241405), pRaf (BioVision Cat# 3504-100, RRID:AB_2060496), pMEK1/2 (Cell Signaling Technology Cat# 9121, RRID:AB_331648), pSMAD5 (Abcam Cat# ab92698, RRID:AB_10561456), SMAD5 (Abcam Cat# ab40771, RRID:AB_777981), pACC (Cell Signaling Technology Cat# 3661, AB_330337), ACC (Cell Signaling Technology Cat# 3662, RRID:AB_2219400), pmTOR(Cell Signaling Technology Cat#2974, RRID: AB_2262884), mTOR (Cell Signaling Technology Cat#2983, RRID: AB_ 2105622), α/β-Tubulin (Cell Signaling Technology Cat#2148, RRID: AB_2288042), GAPDH (GeneTex Cat# GTX100118, RRID:AB_1080976), β-actin (GeneTex Cat# GTX109639, RRID:AB_1949572) and ubiquitin (Cell Signaling Technology Cat# 3936, RRID:AB_331292) were purchased from Cell Signaling (Danvers, Massachusetts, USA), Abcam (Cambridge, Massachusetts, USA), and GeneTex (Irvine, CA, USA).

Techniques: Expressing, Control, Western Blot, Knockdown, shRNA, Phospho-proteomics, Inhibition, Migration, Activity Assay

Journal: bioRxiv

Article Title: Stabilization of AMPK/PFKL/RPIA in the Glycolytic Bodies Transduces IL6/STAT3 Signal in Hepatocarcinogenesis

doi: 10.1101/2024.02.29.582877

Figure Lengend Snippet: ( A ) Treatment with the STAT3 inhibitors nifuroxazide and BBI608 reduces AMPK mRNA levels in PLC5 cells treated with IL6. ( B ) Nifuroxazide and BBI608 treatments block IL6-induced PFKL-upregulated mRNA in PLC5 cells. ( C ) Chromatin immunoprecipitation assays reveal that pSTAT3 directly binds to the promoter regions of AMPK and PFKL in IL6-treated PLC5 cells. ( D ) Inhibition of STAT3 by nifuroxazide and BBI608 diminishes the IL6-mediated increase in PFKL protein levels, as shown by Western blot analysis. The lower panel quantifies the western blot results for PFKL, STAT3, and pSTAT3 protein levels. Red bars represent nifuroxazide treatment, blue bars denote BBI608 treatment, and black bars represent the no treatment control. ( E ) Treatment with the AMPK inhibitor BBI608 reduces the levels of glycolytic and lipogenic metabolites. Red bars represent the BBI608 treatment, while black bars represent the no treatment control. ( F ) Knockdown of PFKL with shPFKL leads to a significant reduction in glycolytic and lipogenic metabolites. Red bars indicate shPFKL, and black bars represent shLuc control. ( G ) Nifuroxazide and BBI608 treatments markedly decreased glucose uptake in PLC5 cells, as indicated by a reduction in ( H ) a tumor xenograft model, detected using FDG nuclear imaging. Arrows indicate the xenograft sites. Quantification of glucose uptake at the xenograft sites is shown. Red bars represent the BBI608 treatment, while black bars denote the no treatment control. ( I ) Targeting STAT3 with nifuroxazide and BBI608 reduces the migration ability of PLC5 cells. Red bars indicate nifuroxazide treatment, blue bars denote BBI608 treatment, and black bars represent the no treatment control. ( J ) PLC5 cells cultured in spheres as cancer stem cells for 10 days under specific conditions. ( K ) PLC5 tumorspheres exhibit increased migration ability compared to parental cells. ( L ) PLC5 tumorspheres display elevated mRNA expression levels of PFKL and AMPK. ( M ) The size of PLC5 tumorspheres can be reduced by STAT3 inhibitors but not by sorafenib. ( N , O ) Knockdown of PFKL and AMPK and suppression of AMPK and STAT3 resulted in a remarkable reduction in mitochondrial respiration and ATP production. Statistical analyses were performed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The specific antibodies against PFKL (Cell Signaling Technology Cat# 8175, RRID:AB_11178807), STAT3 (Cell Signaling Technology Cat# 9132, RRID:AB_331588), pSTAT3 (Cell Signaling Technology Cat# 9130, RRID:AB_330367), AMPK (Cell Signaling Technology Cat# 2603, RRID:AB_490795), pAMPK (Cell Signaling Technology Cat# 5759, RRID:AB_10949320), ERK (GeneTex Cat# GTX59618, RRID:AB_10726211), pERK (Abcam Cat# ab32538, RRID:AB_11156273), RPIA (Abcam Cat# ab67080, RRID:AB_1142656), PK (GeneTex Cat# GTX111536, RRID:AB_1951258), G6P (GeneTex Cat# GTX113203, RRID:AB_2037119), TPI (GeneTex Cat# GTX104618, RRID:AB_1241405), pRaf (BioVision Cat# 3504-100, RRID:AB_2060496), pMEK1/2 (Cell Signaling Technology Cat# 9121, RRID:AB_331648), pSMAD5 (Abcam Cat# ab92698, RRID:AB_10561456), SMAD5 (Abcam Cat# ab40771, RRID:AB_777981), pACC (Cell Signaling Technology Cat# 3661, AB_330337), ACC (Cell Signaling Technology Cat# 3662, RRID:AB_2219400), pmTOR(Cell Signaling Technology Cat#2974, RRID: AB_2262884), mTOR (Cell Signaling Technology Cat#2983, RRID: AB_ 2105622), α/β-Tubulin (Cell Signaling Technology Cat#2148, RRID: AB_2288042), GAPDH (GeneTex Cat# GTX100118, RRID:AB_1080976), β-actin (GeneTex Cat# GTX109639, RRID:AB_1949572) and ubiquitin (Cell Signaling Technology Cat# 3936, RRID:AB_331292) were purchased from Cell Signaling (Danvers, Massachusetts, USA), Abcam (Cambridge, Massachusetts, USA), and GeneTex (Irvine, CA, USA).

Techniques: Blocking Assay, Chromatin Immunoprecipitation, Inhibition, Western Blot, Control, Knockdown, Imaging, Migration, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Stabilization of AMPK/PFKL/RPIA in the Glycolytic Bodies Transduces IL6/STAT3 Signal in Hepatocarcinogenesis

doi: 10.1101/2024.02.29.582877

Figure Lengend Snippet: pSTAT3 increases the transcription of AMPK and PFKL. AMPK stabilizes the PFKL protein level. PFKL specifically stabilizes RPIA. AMPK, PFKL and RPIA are colocalized in the G-body. Inhibiting STAT3 or AMPK can reduce tumor proliferation and metastasis.

Article Snippet: The specific antibodies against PFKL (Cell Signaling Technology Cat# 8175, RRID:AB_11178807), STAT3 (Cell Signaling Technology Cat# 9132, RRID:AB_331588), pSTAT3 (Cell Signaling Technology Cat# 9130, RRID:AB_330367), AMPK (Cell Signaling Technology Cat# 2603, RRID:AB_490795), pAMPK (Cell Signaling Technology Cat# 5759, RRID:AB_10949320), ERK (GeneTex Cat# GTX59618, RRID:AB_10726211), pERK (Abcam Cat# ab32538, RRID:AB_11156273), RPIA (Abcam Cat# ab67080, RRID:AB_1142656), PK (GeneTex Cat# GTX111536, RRID:AB_1951258), G6P (GeneTex Cat# GTX113203, RRID:AB_2037119), TPI (GeneTex Cat# GTX104618, RRID:AB_1241405), pRaf (BioVision Cat# 3504-100, RRID:AB_2060496), pMEK1/2 (Cell Signaling Technology Cat# 9121, RRID:AB_331648), pSMAD5 (Abcam Cat# ab92698, RRID:AB_10561456), SMAD5 (Abcam Cat# ab40771, RRID:AB_777981), pACC (Cell Signaling Technology Cat# 3661, AB_330337), ACC (Cell Signaling Technology Cat# 3662, RRID:AB_2219400), pmTOR(Cell Signaling Technology Cat#2974, RRID: AB_2262884), mTOR (Cell Signaling Technology Cat#2983, RRID: AB_ 2105622), α/β-Tubulin (Cell Signaling Technology Cat#2148, RRID: AB_2288042), GAPDH (GeneTex Cat# GTX100118, RRID:AB_1080976), β-actin (GeneTex Cat# GTX109639, RRID:AB_1949572) and ubiquitin (Cell Signaling Technology Cat# 3936, RRID:AB_331292) were purchased from Cell Signaling (Danvers, Massachusetts, USA), Abcam (Cambridge, Massachusetts, USA), and GeneTex (Irvine, CA, USA).

Techniques: