EMBO/soda-nel huggingface dataset:
CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk (2015)
Figure 1

Deletion of Syk after Tam treatment of mb1‐CreERT2;Sykfl/fl mice

Schematic representation of the targeted mb‐1 locus harboring the CreERT2 construct and the “floxed” Syk locus. The construct is inserted between exons I and IV. The filled rectangles represent the exons of the mb‐1 allele. The open rectangle represents the CreERT2 construct followed by a poly‐adenylation (pA) site. The black circle represents the endogenous pA site of the mb‐1 allele. The “truncated” version of mb‐1 exon I shown here lacks the ATG start codon and is followed by the complete mb‐1 intron I with its splice donor and acceptor sites. Intron I was maintained to provide intron/exon splicing in the transcript and as a source of possible transcription regulatory sequences. The targeted Syk locus was targeted with 2 LoxP sites and has been described in Saijo et al ().
RT‐PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam‐treated mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/fl mice (right lane); the Syk‐ and Hprt‐specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control.
Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/fl (right lane) mice both treated with Tam as described; blots were probed with anti‐Syk and anti‐GAPDH Ab, GAPDH being used as a loading control.
Intracellular flow cytometric analysis for Syk expression in B cells derived from the LN of Syk‐deficient and control mice.
Genomic DNA analysis of Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl mice (as indicated). Upper row: amplification of floxed (fl) and wt (+) alleles. Middle row: amplification of the deleted (d) allele. Lower row: loading control (TC21, a gene unaffected by the deletion of Syk). Established control DNA was used to visualize the individual bands (as indicated).
CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk (2015)
Figure 2

Relative and absolute cell numbers of B‐cell populations in induced mb1‐CreERT2;Sykfl/fl mice

A, B Flow cytometric analysis of B cells from (A) the BM and (B) the SP of mb1‐CreERT2 control (left) and mb1‐CreERT2;Sykfl/fl mice (right) treated with Tam as described in the Materials and Methods section. The BM cells were stained with anti‐IgM and anti‐B220, and the SP cells with anti‐CD19 and anti‐CD93 or anti‐CD23 and anti‐CD21. The gated regions in the dot blots correspond to individual B‐cell populations: (A) Bone marrow: gate P (B220+ IgM−) pro‐/pre‐B cells, gate I (B220lo IgM+) immature B cells, gate M (B220hi IgM+) mature B cells; (B) spleen: gate T (CD19+ CD93+) transitional (T) B cells, gate M (CD19+ CD93−) mature B cells; in CD93− splenic B cells, gate M (CD23hiCD21int CD93−) mature follicular B cells and gate MZ (CD23lo CD21hi CD93−) marginal zone B cells. The numbers in the dot plots indicate the mean relative frequency of cells in the gate.
C–E Statistical analysis of absolute cell numbers per Tam‐treated mouse: filled circles indicating cells obtained from mb1‐CreERT2 control mice and open circles from mb1‐CreERT2;Sykfl/fl mice. (C) Statistical analysis of the absolute cell numbers of pro‐/pre‐, immature and mature recirculating B cells in the BM. (D) Absolute cell numbers of transitional, M and MZ B cells found in the SP. (E) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1‐a and B1‐b. An asterisk (*) marks statistically significant differences (P 0.05), two asterisks (**) indicate P 0.01, P‐values were obtained using two‐tailed Student's t‐test. Cell numbers of four to five mice per group are shown, with each dot representing an individual animal.

Data information: The results are representative of three independent experiments.
CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk (2015)
Figure 3

Effect of syk deletion on B‐cell stimulation

A Flow cytometric analysis of IgM, CD19 and CD23 expression of splenic B cells from Tam‐treated mb1‐CreERT2 control (dashed line) and mb1‐CreERT2;Sykfl/fl mice (solid line).
B Flow cytometric analysis of tyrosine phosphorylation (pY) in splenic B cells from Tam‐treated mb1‐CreERT2 and mb1‐CreERT2;Sykfl/fl mice stimulated with medium (dotted line) or 50 μM pervanadate (dashed and solid lines).
C, D Flow cytometric analysis of the intracellular Ca2+ influx in purified splenic B cells derived from Tam‐treated mice. mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl were treated with (C) 10 μg/ml anti‐Kappa F(ab′)2 fragments or (D) 1 μM ionomycin.

Data information: Data shown are representative of three independent experiments.
CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk (2015)
Figure 4

Mature B cells deficient in Syk are not activated and do not proliferate when stimulated via the BCR

Ex vivo activation assay of splenic mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/fl mice (lower row). Purified splenic B cells were either left unstimulated (medium) or were stimulated with the indicated stimuli. The cells were analyzed by flow cytometry after 24 h. Shown are CD19 versus CD86 dot plots. 7‐AAD was included to distinguish dead from viable cells. The numbers in the quadrants indicate the relative frequencies of cells in the gate.
Ex vivo proliferation assay with mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/fl mice (lower row). The cells were incubated with the indicated stimuli for 90 h.

Data information: Data shown are representative of three independent experiments.
Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance (2019)
Figure 1

Figure 1. Chlorambucil sensitivity of BRCA2-deficient human cells and spheroids.

Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with drugs at the indicated concentrations for six days.

Human spheroids established from BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) DLD1 cells, were incubated with 1.25 µM olaparib or 0.5 µM chlorambucil over the indicated period of time.

Data information: (A, B) Graphs represent average values obtained from three independent experiments, each performed in triplicate. Error bars represent SEM.
Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance (2019)
Figure 2

Figure 2. Chlorambucil sensitivity of BRCA1-deficient human and mouse cells, including those that have acquired olaparib resistance.

Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) human RPE1-hTERT and TP53-deleted cells treated with drugs at the indicated concentrations for six days.

Dose-dependent viability assays of Brca1+/+ and Brca1-/- mouse mammary tumour-derived cell lines treated with drugs at the indicated concentrations for six days.

Data information: (A, B, C) Graphs represent average values obtained from three independent experiments, each performed in triplicate. Error bars represent SEM.
Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance (2019)
Figure 3

Figure 3. Chlorambucil sensitivity of BRCA2-deficient human tumour-derived cell lines, including those that have acquired cisplatin resistance.

Dose-dependent viability assays of BRCA2-deficient (Capan-1) or -proficient (MIA PaCa-2) human pancreatic carcinoma-derived cells treated with drugs at the indicated concentrations for six days.

Dose-dependent viability assays of BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells treated with drugs at the indicated concentrations for six days.

BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells were infected with lentiviruses expressing control or CHD4 shRNAs, followed by selection with puromycin for 72 hours. Dose-dependent viability assays were performed on cells treated with drugs at the indicated concentrations for six days.

Data information: (A, B) Graphs represent average values obtained from three independent experiments, each performed in triplicate. Error bars represent SEM. (C) Error bars represent SEM of three technical replicates.
Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance (2019)
Figure 4

Figure 4. DNA damage responses to chlorambucil and cisplatin in human cells.

BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells were incubated with 1 µM chlorambucil (Chl). Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. GAPDH was used as a loading control.

Quantification of chromosome aberrations and chromatid/chromosome break frequencies in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h. Data were obtained from three independent experiments and normalised to untreated controls. A minimum of 60 Giemsa-stained metaphases were analysed for each sample. Cis, cisplatin; Chl, chlorambucil.

Quantification of G2/M cell frequency relative to solvent control, using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours. Cis, cisplatin; Chl, chlorambucil.

Human H1299 cells were treated with control (CTR) or indicated siRNAs two days before drugs were added to the media for dose-dependent viability assays. Cell extracts prepared at the time of drug addition were immunoblotted as indicated. SMC1 was used as a loading control.

Data information: (B) Whiskers indicate 10-90 percentile and red bars indicate mean frequencies of breaks. P values were calculated using the Mann-Whitney test. ****, P < 0.0001. (C) Error bars represent SEM of three independent experiments. P values were calculated using an unpaired two-tailed t-test. **, P ≤ 0.01. NS, P > 0.5. (D, E) Graphs represent average values obtained from three independent experiments, each performed in triplicate. Error bars represent SEM. Exact P-values are included in Appendix Table S1.
Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance (2019)
Figure 5

Figure 5. Chlorambucil impairs growth of BRCA2-deficient tumours and PDTXs.

Nude mice (nu/nu) were injected subcutaneously with 5 x 106 human DLD1 cells, BRCA2-proficient (A) or BRCA2-deficient (B). Tumour-bearing mice were treated with 3 mg/kg daily chlorambucil administered intraperitoneally (i.p.) for a total of ten days. Tumour weight was determined on the indicated days after initiation of the treatment.

PDTCs derived from breast cancer samples as previously described (Bruna et al, 2016) were treated with chlorambucil at the indicated doses. Cell survival is represented relative to DMSO control. AB521, ER-negative tumour, no BRCA1 alteration; STG201, tumour with BRCA1 promoter methylation and loss of BRCA1 expression; VHIO179, tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant); http://caldaslab.cruk.cam.ac.uk/bcape/.

CB17/SCID mice were injected intramuscularly with 5 x 106 human BRCA2-deficient HCT116 cells. Tumour-bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally (i.p.), or talazoparib administered orally (o.s.) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment (day 1). Scale bar, 40 µm.

Data information: (A, B) Each experimental group included n = 4 mice. Error bars represent SEM. (C) Error bars represent SEM. (D) Each experimental group included n = 5 mice. Error bars represent SEM. Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining. P values were calculated using an unpaired two-tailed t-test. ****, P < 0.0001; ***, P < 0.001. Exact P-values are included in Appendix Table S1.
Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance (2019)
Figure 6

Figure 6. Chlorambucil vs cisplatin in vitro and in vivo toxicity.

Wild type Balb/c mice were injected intra-peritoneally with solvent (daily) or 3 mg/kg chlorambucil (daily for 5 days) or 3.3 mg/kg cisplatin (daily for 3 days). Uptake of the apoptosis tracer 99mTc-Duramycin 2 h after intra-venous injection was quantified in selected organs using SPECT imaging in the indicated organs. Representative maximum intensity partial projections showing tracer distribution are shown.

Immunohistochemical analyses of γH2AX staining in organs from mice treated as in A. Scale bar, 25 µm.

Data information: (A) Each experimental group included n = 5 mice. P values were calculated using 1way ANOVA followed by Tukey's multiple comparisons test. ****, P < 0.0001; *, P < 0.05. (B) Organs from n = 3 mice were analysed for each treatment. P values were calculated using 1way ANOVA followed by Tukey's multiple comparisons test. ****, P < 0.0001; *, P < 0.05. Exact P-values are included in Appendix Table S1.
Non-canonical phosphoglycerate dehydrogenase activity promotes liver cancer growth via mitochondrial translation and respiratory metabolism (2022)
Figure 1

Figure 1. PHGDH localizes to the mitochondrial inner membrane in liver cancer cells.

A Western blot analysis of the localization of endogenous PHGDH in PLC, HepG2, and THLE3 cells. Lamin B1, Tubulin, and Tom20 were used as markers for nuclear (Nuc), cytosolic (Cyto), and mitochondrial (Mito) proteins, respectively.

B Western blot analysis of the localization of endogenous PHGDH in tumour tissues and adjacent normal tissues. Lamin B1, GAPDH, and Tom20 were used as markers for Nuc, Cyto, and Mito proteins, respectively.

C Western blot analysis of the localization of endogenous and exogenous Flag-tagged PHGDH in PLC cells. Lamin B1, Tubulin, and Tom20 were used as markers for Nuc, Cyto, and Mito proteins, respectively.

D Immunofluorescence images showing colocalization of Flag-PHGDH (green) and mitochondria (red) acquired with a laser confocal microscope system in PLC cells. Nuclei were stained with DAPI (blue), and mitochondria were stained with MitoTracker Red. Scale bar, 10 μm (left panel), 2 μm (right panel).

E The PHGDH structure is displayed with the N-terminus (substrate binging domain 1, SBD1), nucleotide binding domain (NBD), substrate binging domain 2 (SBD2), and the C-terminus (regulatory domain, RD). The PHGDH∆SBD1 and PHGDH∆RD mutant variants lack the SBD1 and RD, respectively.

F Subcellular localization of PHGDH was determined by western blot in PLC cells overexpressing of Flag-tagged wild-type PHGDH, PHGDH∆SBD1, or PHGDH∆RD mutants. WCL: whole-cell lysates, Mito: mitochondria. Tom20 was used as loading control.

G Proteinase K protection assays were performed on purified mitochondria isolated from PLC and HepG2 cells. The outer mitochondrial membrane (OMM) protein Tom20, the inner mitochondrial membrane (IMM) protein ANT2, and the mitochondrial matrix (MM) protein TFAM were used as markers of the OMM, IMM, and MM, respectively.

H Purified mitochondria of PLC or HepG2 cells were incubated with Na2CO3 (pH 11.5) for 30 min at 4 ℃. The soluble component (Supernatant/Sup.) and membrane component (Pellet) were separated by centrifugation prior to western blot analysis. COX2, ATP8, and ANT2 are markers for the membrane compotent; CYTC and TFAM are markers for the soluble component.

Source data are available online for this figure.
Non-canonical phosphoglycerate dehydrogenase activity promotes liver cancer growth via mitochondrial translation and respiratory metabolism (2022)
Figure 2

Figure 2. PHGDH promotes the translation of mtDNA-encoded proteins.

A The oxygen consumption rate (OCR) was measured by successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot) in PLC cells infected with viruses expressing nontargeting control (NTC) shRNA or shPHGDH. Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.

B Western blot analysis of mtDNA-encoded proteins in PLC cells stably expressing NTC or shPHGDH and empty vector (EV) or PLVX-PHGDH. β-Actin was used as the loading control.

C PLC cells with endogenous PHGDH knockdown were further infected with viruses expressing EV or PLVX-PHGDH prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.

D PLC cells expressing NTC or shPHGDH were treated with 100 μg/ml cycloheximide (CHX) for 20 min prior to puromycin treatment for 15 min. Purified mitochondrial lysates were subjected to a SUnSET assay with an anti-puromycin antibody to measure the actively translated polypeptide chains. Western blot analysis of PHGDH confirmed the knockdown efficiency of the PHGDH shRNAs. β-Actin was used as the loading control.

E SUnSET assays were performed in the indicated cell lines using whole-cell lysates.

F PLC cells with or without PLVX-Flag-PHGDH overexpression were treated with DMSO or 50 μM chloramphenicol (CAP) in combination with 50 μM thiamphenicol (TAP) for 24 h prior to SUnSET assays. The assays were performed using whole-cell lysate.

G PLC cells with or without PLVX-PHGDH overexpression were treated with DMSO or 50 μM CAP in combination with 50 μM TAP for 24 h prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.

H The translation efficiency of mtDNA-encoded proteins was determined by a SUnSET assay in PLC cells with endogenous PHGDH knockdown that were further infected with viruses expressing EV, wild-type PHGDH, or the catalytically inactive PHGDH mutant PHGDHR236E as indicated. β-Actin was used as the loading control.

I mtDNA-encoded proteins were analysed by western blotting in PLC cells overexpressing EV, wild-type PHGDH, or PHGDHR236E. β-Actin was used as the loading control.

Data information: n = 3 independent experiments and the data are presented as the mean ± SD values in (A). Statistical significance was determined by one-way ANOVA (A). *P < 0.05 as compared to NTC group.

Source data are available online for this figure.
Non-canonical phosphoglycerate dehydrogenase activity promotes liver cancer growth via mitochondrial translation and respiratory metabolism (2022)
Figure 3

Figure 3. mtEFG2 and ANT2 are required for PHGDH-mediated mitochondrial translation.

A HEK293T cells were transfected with Flag-tagged PHGDH in combination with HA-tagged EV, mtIF2, mtIF3, mtEFTu, mtEFG1, mtEFG2, or mtRRF. Co-immunoprecipitation (Co-IP) was performed with an anti-Flag antibody prior to western blot analysis.

B HEK293T cells were transfected with HA-tagged PHGDHWT or PHGDHR236E in combination with Flag-EV or Flag-mtEFG2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.

C HEK293T cells were transfected with HA-tagged PHGDHWT or PHGDHR236E in combination with Flag-EV or Flag-ANT2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.

D HEK293T cells were transfected with HA-mtEFG2 in combination with Flag-EV or Flag-ANT2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.

E GST pull-down of His-PHGDHWT and His-PHGDHR236E by GST-ANT2 using proteins purified in E. coli, followed by western blot analysis with anti-His and anti-GST antibodies.

F GST pull-down of His-mtEFG2 by GST-ANT2 using proteins purified in E. coli, followed by western blot analysis with anti-His and anti-GST antibodies.

G PLC cells stably expressing Flag-mtEFG2 and HA-PHGDH were further infected with viruses expressing NTC or shANT2, and then cell lysates were immunoprecipitated with an anti-Flag antibody or IgG prior to western blot analysis.

H PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shANT2. The translation efficiency was measured by a SUnSET assay.

I PLC cells overexpressing PLVX-PHGDH were further infected with viruses expressing NTC or shANT2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.

J PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shmtEFG2. The translation efficiency was measured by a SUnSET assay.

K PLC cells overexpressing pSin-3xFlag-PHGDH were further infected with viruses expressing NTC or shmtEFG2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.

Source data are available online for this figure.
Non-canonical phosphoglycerate dehydrogenase activity promotes liver cancer growth via mitochondrial translation and respiratory metabolism (2022)
Figure 4

Figure 4. PHGDH promotes mtEFG2-mediated mitochondrial ribosome recycling in cancer cells.

A HEK293T cells were transfected with HA-mtEFG2 in combination with Flag-EV or Flag-mtRRF, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.

B PLC cells stably expressing Flag-mtEFG2 and HA-mtRRF were further infected with viruses expressing NTC or shPHGDH, and then cell lysates were immunoprecipitated with anti-Flag antibody or IgG prior to western blot analysis.

C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. The translation efficiency was measured by a SUnSET assay.

D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. mtDNA-encoded proteins were analysed by western blot. β-Actin was used as the loading control.

E PLC cell lysates were immunoprecipitated with an anti-MRPL44 antibody or IgG prior to western blot analysis of MRPL44, MRPL46, MRPL48, MRPS15, MRPS16, and MRPS35.

F Binding of mtDNA-encoded mRNA by endogenous MRPL44 (representing mitochondrial ribosomes) was evaluated by RNA immunoprecipitation (RIP) in PLC cells with PHGDH knockdown.

G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient. After centrifugation, all fractions were subjected to western blot analysis of MRPS35, MRPL44, and MRPL48 proteins. RNA concentrations of ND6 were measured.

Data information: n = 3 independent experiments in (F), the data are presented as the mean ± SD values. The average of three independent experiments is shown in (G).

Source data are available online for this figure.