EMBO/soda-nel huggingface dataset:
MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability (2013)
figf4

(a) Transcriptional levels of hlh-30 and the mxl-3-dependent lipl genes measured in 5 h fasted wild-type (WT) or hlh-30(tm1978) young-adult worms depicted as mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms show that hlh-30 is induced following fasting and HLH-30 induces lysosomal lipolysis.
N = 4independent experiments for lipl data,
n = 2 independent experiments for hlh-30 mRNA. HLH-30 deficiency fully abrogates lipl-2, 3 and 5 (P0.0001), and impairs lipl-1 (P0.01) transcriptional activation following fasting. (b) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults. Mean+s.e.m. of three independent experiments shows that inhibition of TOR is sufficient to induce hlh-30 transcription. (c) L3 animals carrying the rescuing construct hlh-30P::HLH-30::mGFP::hlh-30 3′UTR (mGFP, monomeric GFP, and UTR, untranslated region), well fed or fasted for 8 h show that HLH-30 is enriched in intestinal nuclei of fasted worms (exposure time: well fed, 500 ms; fasted, 100 ms). (d) ORO staining of wild-type or hlh-30(tm1978) young adults fasted for 8 h reveals that hlh-30 is required for optimal lipid mobilization on fasting. Mean percentages+s.e.m. are shown below relative to well-fed worms treated in parallel,
n = 3 independent experiments. (e) mxl-3 and hlh-30 transcriptional levels in wild-type,
hlh-30(tm1978) and mxl-3(ok1947) mutants in the basal and fasted states show that
mxl-3 transcription is hlh-30 independent, and hlh-30 transcription is mxl-3 independent. Expression is presented relative to wild-type fed animals as mean ddCt+s.e.m. No significant differences relative to wild type in the same feeding state were observed,
n = 3 independent experiments. (f) Representative ChIP-qPCR analysis of well-fed and 6 h fasted mixed stage worms expressing HLH-30::GFP presented as in Fig. 3d shows that HLH-30 occupies the lipl promoters during early fasting. See raw data of two independent experiments in Supplementary Table S9. (g) Transcriptional levels of the mxl-3-dependent lipl genes in wild-type, mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant young-adult worms show that hlh-30 suppresses the constitutive-induction-of-the-lipl-genes phenotype of mxl-3 mutant animals (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms).
n = 3 independent experiments.
MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability (2013)
figf5

(a) LRO (lysosome-related organelle) live Nile red-stained images of hlh-30(tm1978) and wild-type (WT) L3 larvae starved for 60 h show that hlh-30 is required for the expansion of the lysosomal compartment following fasting. Quantification as mean±s.e.m. relative to fasted wild-type worms is also presented;
n = 3 independent experiments. (b) Transcriptional levels of the autophagy genes lgg-1, lgg-2 and atg-16.2 in fasted wild-type and hlh-30(tm1978) young-adult worms show that transcriptional activation of autophagy following fasting is hlh-30 dependent (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms).
n = 3 independent experiments. (c) The mean percentage (+ s.d.) of L1 larvae alive after 48 h fasting in minimum media shows that hlh-30 (tm1978) mutant animals are sensitive to starvation.
n = 3 independent experiments. For L4 survival see Supplementary Fig. S6g.
MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability (2013)
figf6

(a) Expression analyses of the liver of C57BL/6J 9-week females fasted overnight (ON) for 10 h relative to siblings feeding
ad libitum show that LipA (mouse lysosomal acid lipase), Map1lc3a (mammalian lgg-1/lgg-2) and Tfeb (mammalian hlh-30) but not Max (mammalian mxl-3) are transcriptionally linked to nutrients in the mouse liver (median+s.e.m. fold change; ddCt). Levels of expression were normalized to ActB and
Cog2 as internal controls; all but Max differences are significant (P0.05),
n = 3 independent experiments. Cyp4a14 is a positive control. (b) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2, 4 or 8 h, compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media, show that LAL (human lysosomal acid lipase), MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes, and that LAL and
MAP1LC3A induction under nutrient deprivation are TFEB dependent (median dCt+s.e.m.). Levels of expression were normalized to ACTB as internal control; all butMAX differences are significant (P0.05),
n = 3 independent experiments. IGFBP is a positive control. EBSS, Earles’s balanced salt solution.
MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability (2013)
figf7

(a) Wild-type (WT),
mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant were used in longevity epistasis analyses, revealing that hlh-30 suppresses the mxl-3 extended lifespan phenotype. Animals were incubated at 20 °C and transferred every other day until cessation of reproduction. The cumulative survival curve is depicted. Kaplan–Meier statistics, calculated using SSPS 17, are also shown. (b) Live Nile red quantification of wild-type and lipl-1lipl-3 double mutant worms shows that lipl-1 and lipl-3 contribute to the clearance of lipids from the endocytic pathway. Mean+s.e.m. signal intensity is shown as percentage relative to wild type; significant differences are indicated;
n = 4 independent experiments. (c,d) Animals overexpressing LIPL-1 or LIPL-3 were incubated at 25 °C and transferred every other day until cessation of reproduction. Data presented as in Fig. 7a show that activated lysosomal lipolysis extends C. elegans lifespan.
a Survival presented as mean lifespan (LS) ± s.e.m. b Number of uncensored animals (animals that crawled off the plate, bagged, exploded or became contaminated were censored).
Nuclease-independent functions of RAG1 direct distinct DNA damage responses in B cells (2022)
Figure 1

Figure 1. RAG DSBs at Igk and Igh activate cDDR and ncDDR.

A-D. Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (A) DSBs quantified by qPCR analysis of Igk (Jκ1) genomic DNA. Schematic shows germline (GL) Igk locus, unrepaired Jκ1 coding end (Cut) and primer location. Results are normalized to Rag1-/-:Bcl2 abl pre-B cells, which do not generate RAG DSBs and have only germline Igk DNA. Loss of Igk germline product is representative of DSB generation. (B) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified. (C) Western blot analysis of KAP1 phosphorylation (p-KAP1), NF-κB2 (p100 and p52) and PIM2. GAPDH is shown as loading control. Quantitation in Fig EV1B. (D) Cd40 and Pim2 mRNA expression.

E-H. Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (E) DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in A with results normalized to empty vector control, which has only germline Igh DNA. (F) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified. (G) Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control. Quantitation in Fig EV1D. (H) Cd40 and Pim2 mRNA expression.

Data information: In A, B, D, E, F, and H, data are mean ± SEM for three biological replicates. In C and G, data are representative of three biological replicates. Significance by Student's t-test: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001
Nuclease-independent functions of RAG1 direct distinct DNA damage responses in B cells (2022)
Figure 2

Figure 2. Cas9 DSBs at non-Ig genes do not activate ncDDR.

A. Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with (+) or without (-) 2 μM doxycycline (Dox) to induce Cas9 as indicated. Western blot shows FLAG-Cas9 and GAPDH (loading control).

B-F. Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with doxycycline as in A and imatinib to trigger cell cycle arrest for 24 h. Cells were then transfected with indicated gRNA and maintained in doxycycline and imatinib. All analyses were completed 48 h after gRNA transfection. DSBs quantified by qPCR analysis of Eb (B) and Gapdh (C) genomic DNA. Results are normalized to cells without gRNA (-). Schematic shows germline (GL) locus, unrepaired cut end (Cut) and location of gRNA and primers. Loss of germline product is representative of DSB generation. (D) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified. (E) Western blot analysis of FLAG-tagged Cas9 or RAG1 (as indicated), p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control. Responses to RAG DSBs in Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells transduced with empty vector or vector expressing RAG1 RAG DSBs from Fig 1G are included for direct comparison. # denotes band for FLAG-Cas9. * denotes band for FLAG-RAG1. Quantitation in Fig EV2D. (F) Cd40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.

Data information: In B, C, D, and F, data are mean ± SEM for three biological replicates. In A and E, data are representative of three biological replicates. Significance by Student's t-test: ns = not significant, *p ≤ 0.05
Nuclease-independent functions of RAG1 direct distinct DNA damage responses in B cells (2022)
Figure 3

Figure 3. Cas9 DSBs at Ig genes do not activate ncDDR.

Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with imatinib and doxycycline as in Fig 2 then transfected with Jκ1, JH1, JHD1-1 or both JH1 + JHD1-1 gRNAs as indicated. Following transfection, cells were maintained in doxycycline and imatinib with all analyses completed 48 h after gRNA transfection.

A-B. DSBs quantified by qPCR analysis of Jκ1 (A) and JH1 and JHD1-1 (B) genomic DNA. Schematics show germline (GL) locus and location of respective gRNAs and primers. PCR is normalized to cells without gRNA (-). Loss of germline product is representative of DSB generation.

C. Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.

D. Western blot analysis of FLAG-Cas9, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control. Quantitation in Fig EV3E.

E. CD40 and Pim2 mRNA expression. Blue dashed line indicates mRNA expression in response to RAG DSBs from Fig 1H and is included for comparison.

Data information: In A, B, C, and E, data are mean ± SE for three biological replicates. In D, data are representative of three biological replicates. Significance by Student's t-test: ns = not significant, *p ≤ 0.05, **p ≤ 0.01
Nuclease-independent functions of RAG1 direct distinct DNA damage responses in B cells (2022)
Figure 4

Figure 4. Non-core regions of RAG1 are necessary for activation of ncDDR.

Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were retrovirally transduced with empty vector or vector expressing indicated RAG1 variants then treated with imatinib for 48 h.

A. Schematic of RAG1 variants. Figure not to scale.

B. DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in Fig 1E with results normalized to empty vector control, which has only germline Igh DNA. There is no statistically significant difference in DSBs generated by cRAG1 or RAG1P326G compared to full-length RAG1.

C. Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.

D. Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control. Quantitation in Fig EV5D.

E. Cd40 and Pim2 mRNA expression.

Data information: In B, C, and E, data are mean ± SEM for three biological replicates. In D, data are representative of three biological replicates. Significance by Student's t-test: ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

EXPANDED VIEW FIGURE LEGENDS
Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal gene transfer (2016)
Figure 3

Figure 3. Functionality of NILV-S/MAR-engineered CD19 CAR T cells. a) Schematic drawing of the lentiviral vectors used. Abbreviations as in Figure 1; EF1α prom: the human elongation factor-1α gene promoter; CD19CAR: chimeric antigen receptor (CAR) targeting the pan-B cell marker CD19 (for detailed information see Materials and Methods). b) A schematic timeline of the experimental setting. c) Percentage of CD19CAR expression on CD3+ T cells from each independent donor. d) Histograms of CD19 CAR expression on T cells form one representative donor before and after expansion. e) CD107a expression on transduced T cells (CD3+) after exposure to CD19+ target cells (Daudi) for 24 h (n=6). f) IFN-γ production in the supernatant was analyzed by ELISA after 24 h of exposure of transduced T cells to target cells (n=6). g) Relative viability of target cells (CD19+ Daudi), as an indication of T cell killing efficacy, were measured at different effector T cell to target cell ratio (E:T ratio) and normalized to the signal from target cells alone (n=6). h) Transduced T cells were labeled with a fluorescent cell membrane dye and co-cultured with CD19+ Daudi cells for 4 days. Dye dilution upon T cell division was monitored by flow cytometry and representative histograms are shown. i-m) Mice were treated by intravenous injection of NILV-S/MAR(Mock), LV(CD19CAR) and NILV-S/MAR(CD19CAR)-transduced T cells (8x106 cells/injection) at days 18, 21, 24 after tumor implantation. Tumor growth was monitored by caliper measurements. i-k) Tumor volume of individual mice in each treatment groups are presented. l) The average tumor volume of each treatment group is presented and the values on day 37 were statistically compared (n=12 in LV group, n=9 in NILV-S/MAR group and n=11 in Mock control group). m) The survival of mice is shown as Kaplan-Meier curves. n-p) Representative images showing infiltration of adoptively transferred human T cells into tumors. T cells were visualized by staining for human CD3 (red) and nucleus (blue). q) Quantification of CD3+ T cells from each treatment group is presented (n=5).

Data information: Data are presented as mean±SEM and in some cases results from each donor are presented. Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparisons test while the Kaplan-Meier survival curves were compared using log-rank test with Bonferroni correction. n.s. p≥0.05; * P<0.05; *** P<0.001; **** P<0.0001. Exact P-values between all groups from each test are reported in Appendix Table S1.
An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs (2016)
Figure 1

Figure 1: Effect of Tm1gs on oskar transport.

(A, B) Localization of oskar mRNA in wild-type (A) and in Tm1eg9/Tm1eg9 (B) egg-chambers.

(C) Kymograph of oskMS2-GFP mRNPs (cyan) travelling along polarity marked MTs (red, EB1 protein) in an ex vivo ooplasmic preparation. White dashed arrow shows the growing plus tip of the MT. Scale bars represent 1 s and 1 µm, respectively.

(D) Distribution of oskMS2-GFP mRNP runs towards plus (white) and minus ends (grey). Numbers within the bars indicate number of runs. P value of Chi2 test against wild-type is indicated above each bar.

(E, F) oskar mRNA (green) and Oskar protein (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers expressing oskarMS2(6x) (E) or oskarMS2(6x) and Khc401-MCP (F).

(G) oskar mRNA (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers rescued with EmGFP-Tm1-I (green). Scale bars represent 50 µm.
An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs (2016)
Figure 2

Figure 2: Composition of oskar mRNPs ex vivo.

(A-B”) Colocalization of oskMS2-mCherry (red, A, B) (A’, B’) with Khc-EGFP (green, A) (A’) or with EmGFP-Tm1-I (green B) (B’) in ex vivo ooplasmic preparations.

(C) Fraction of oskMS2-MCP-mCherry mRNPs located non-randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations. MCP indicates MCP-EGFP which, like MCP-mCherry, can bind to MS2 loops. Staufen (Stau) is a dsRNA binding protein and bona fide partner of oskar mRNA (St Johnston et al., 1991; St Johnston et al., 1992). All values are significantly different from zero (p<10-3, one sample t-test).

(D) Fraction of oskMS2-mCherry mRNPs co-localizing (max. 200 nm) non-randomly with Khc-EGFP particles in wild-type and in Tm1eg1/Tm1eg9 ooplasms in presence of two (white) or one (grey) copy of endogenous Khc.

(C, D) P values of two sample t-tests are indicated above the relevant bar pairs. Numbers indicate the number of particle clusters (160 mRNPs in each) and the number of preparations (in brackets) analysed. Error bars represent 95% confidence intervals.
An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs (2016)
Figure 3

Figure 3: Dynamic composition of oskar mRNPs ex vivo

(A, B) Kymographs of oskMS2-GFP mRNPs (green) associated with Khc-mKate2 (A, red) and mCherry-Tm1-I/C (B, red) ex vivo. Arrows indicate motile RNPs in stable complex with Khc (A) or Tm1-I/C (B), the arrowheads point to non-motile oskMS2-RNPs showing no obvious accumulation of the tagged protein. Note that mCherry-Tm1-I was exposed twice as long as Khc-mKate to obtain comparable red fluorescence signals. Scale bars represent 1 µm and 1 second, respectively.

(C-E) Relative Khc-mKate2 (C), mCherry-Tm1-I/C (D) and GFP-Staufen (E) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.

(F) Example kymograph of Khc molecules (red) associating with an oskMS2-GFP mRNP (green) before and during its run. White arrowheads indicate two association events, yellow arrow indicates the onset of motility. Scale bars represent 5 µm and 1 second, respectively.

(G) Frequency of Khc-mKate2 appearance on motile (white), before (motility-primed, checked) and after (dotted) the onset of motility, and non-motile (grey) oskMS2-GFP trajectories. Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed. Indicated P values show results of pairwise Fisher’s exact test. The Khc association frequency observed on RNPs before (checked) and during (dotted) their motility is not significantly different from wild-type motile RNP controls (p>0.01).
An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs (2016)
Figure 4

Figure 4: Tm1-I/C binds RNA and Khc

(A) Western blots of oskar mRNP components (Staufen, Bruno and Y14) and motor associated proteins (Khc, BicD, Dic) co-immunoprecipitated with EmGFP-Tm1-I from ovarian lysates. Protein marker bands and their molecular weight in kDa are indicated.

(B) Signal developed with oligo(dT)25-TexasRed probes on GFP-Trap_M beads binding EmGFP-Tm1-I or FlagMycGFP after denaturing washes. 594 nm reflection marks bead boundaries.

(B’) Quantification of mean oligo(dT) signal measured on beads.

(C) qRT-PCR of EmGFP-Tm1-I and control Flag-Myc-GFP and bead alone (w1118) bound oskar and gapdh RNAs after stringent washes (mean ± s.e.m., N = 3).

(D) Images of beads binding to EmGFP-Tm1-I (green) and DIG labelled in vitro transcribed RNA fragments (magenta).

(E, F) Mean DIG-Cy5 fluorescence measured on beads capturing the RNA fragment, with or without UV cross-linking, indicated below the charts. P values of pairwise Mann-Whitney U tests are indicated. In panel F, none of the non-cross-linked samples differ significantly from the no RNA control (p>0.05). Numbers below the plots indicate the number of beads analysed.
An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs (2016)
Figure 5

Figure 5: Composition of oskar mRNPs in situ

(A, B, C) Confocal image of a Tm1eg9 homozygous egg-chamber expressing EmGFP-Tm1-I (green) and Khc-mKate2 (magenta). oskar mRNA labelled with osk1-5 FIT probes(Hovelmann et al., 2014) is in yellow.

(B’, C’) oskar mRNPs co-localizing with both EmGFP-Tm1-I and Khc-mKate2. Colors indicate the maximal co-localization distance (C’). Panels B-C’ represent the boxed regions in panel A.

(D, E) Localization of Khc-mKate2 (green) and oskar mRNA (magenta) in wild-type and Tm1gs mutant nurse cells.

(F, G) Fraction of oskar mRNPs co-localizing with Khc-mKate (dark grey), EmGFP-Tm1-I (white), or both of these proteins (light grey) in the oocyte (F) or in the nurse cells (G) (max. colocalization distance is 250 nm). None of the values are significantly different from each other (one-way ANOVA, p>10-3). Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent (see Fig EV3C and D). Significance of the observed co-localization values versus the expected values are shown.

(H) Fraction of oskar mRNPs co-localizing with Khc-mKate in wild-type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled (as indicated above the graph). P values of pair-wise t-tests are indicated.

(I) Fraction of oskar mRNPs co-localizing with free mCherry in wild-type nurse cells used as negative control. The measured fraction (~1.6%) is significantly different from zero (one sample t-test). All other measured co-localization values are significantly different form this negative control (p<0.001, one-way ANOVA).

(F-I) Numbers indicate the number of particle clusters (100 oskar mRNPs in each) and the number of egg-chambers (in brackets) analysed. Error bars represent 95% confidence intervals. All values (F-I) are significantly different from zero (p<10-3, one sample t-test).

Scale bars represent 20 µm (A) 5 µm (B, C, E).