EMBO/soda-nel huggingface dataset:
IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation. (2022)
Figure 1

Fig. 1: IL-21 promotes early B cell expansion.

(A) Experimental setup to study the role of IL-21R in early B cell expansion. WT and Il21r-/- SWHEL B cells were transferred together with OTII T cells into recipient mice followed by ip immunization with alum adjuvanted HEL2xOVApep.

(B) Flow cytometry analysis of WT to Il21r-/- splenic SWHEL B cells over time.

(C) WT to Il21r-/- SWHEL B cell ratio within CTV division peaks.

(D) Representative flow cytometry plot of SWHEL B cell CTV profile on day 3.5.

(E) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks.

Data information: Data in (B) is representative of 8-9 biological replicates (n = 8-9) pooled from two independent experiments with statistical analysis by one-way ANOVA with Tukey's post-test. ** p ≤ 0.01; **** p ≤ 0.0001. Data in C-E show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.
IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation. (2022)
Figure 2

Fig. 2: IL-21 increases cell cycle speed and rate of re-entry.

(A) Experimental setup to study cell cycle progression of SWHEL B cells using BrdU. injection.

(B) Flow cytometry analysis showing WT to Il21r-/- SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU.

(C) Schematic depiction of BrdU and DNA content (7AAD) analysis by flow cytometry. At 4 hours, BrdU positive cells with 2N DNA content mark those that have finished the cell cycle, whereas at 10 hours 2N DNA content identifies cells that have not yet entered the subsequent cell cycle.

(D) Ratio of the proportion of BrdU positive WT and Il21r-/- SWHEL B cells with 2N DNA content 4, 10 and 12 hours post BrdU pulse on day 3.5 post immunization.

Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.
IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation. (2022)
Figure 3

Fig. 3: IL-21 directly promotes cell cycle entry.

(A) Experimental setup of to study the immediate consequences of an in vivo IL-21 pulse on SWHEL B cell proliferation.

(B) Schematic depiction of BrdU and 7AAD analysis by flow cytometry. Cells that had not already been in or were about to enter S phase at the time of IL-21 treatment were identified as being BrdU negative. > 2N DNA content among BrdU negative cells marked those that have entered the cell cycle following saline or IL-21 treatment.

(C) Rate of de novo cell cycle entry 10 hours post in vivo IL-21 (2 µg) or saline pulse. The ratio of WT to Il21r-/- cells not in S/G2 at the time of pulse (BrdU- cells) and containing >2N DNA content 10 hours after pulse is shown.

Data information: Data are representative of 10-12 biological replicates (n = 10-12) from two independent experiments. Statistical analysis by one-sample t test. * p ≤ 0.05.
IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation. (2022)
Figure 4

Fig. 4: IL-21 synergizes with BCR and CD40 to promote AKT and S6 phosphorylation.

(A-E) Phosphoflow analysis of naïve WT or Il21r-/- B cells following in vitro culture for 3 hours with or without IL-21 (20 ng/mL) and/or in the presence of BCR cross-linking (biotinylated anti-Igκ + anti-Igλ and avidin-mediated cross-linking) or agonistic anti-CD40. (A) Exemplary p-AKT (S473) staining and (B) quantification of p-AKT median fluorescence intensity (MedFI). (C) Exemplary p-S6 (Ser235/236) staining and (D) quantification of frequency of p-S6 positive cells and (E) p-S6 median fluorescence intensity (MedFI) of p-S6 positive cells.

(F, G) In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (F) Exemplary CTV division peaks. (G) Quantification of the proportion SWHEL B that had divided and thus diluted CTV.

Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by one-way ANOVA with Tukey's post-test (D), t test (B, E) or two-way ANOVA with correction for multiple comparison using Šidák method (G). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation. (2022)
Figure 5

Fig. 5: IL-21 amplifies B cell recruitment into the GC.

(A) Experimental setup to study affinity-dependent effects of IL-21 on B cell recruitment.

(B, C) Analysis of splenic OTII Tfh cells by flow cytometry. (B) Tfh cell count and (C) Proportion of Il21Gfp/+ OTII Tfh cells transcribing the Il21 locus and thus expressing GFP.

(D-G) Analysis of splenic WT and Il21r-/- SWHEL B cells by flow cytometry. (D) CTV profile of and (E) absolute cell count in pooled CTV division peaks (0-4, 5-8, 9-11+). (F) Cell surface expression (median fluorescence intensity, MedFI) of CD38, IgD and FAS across individual CTV division peaks. (G) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression.

Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. D, F and G show concatenated data representative of 3-5 biological replicates. Statistical analysis by one-way ANOVA with Tukey's post-test (B, C). Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm-Šídák method. * p ≤ 0.05; ** p ≤ 0.01.
IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation. (2022)
Figure 6

Fig. 6: High and low affinity B cell responses are amplified by IL-21.

Experimental setup as in Fig. 5A but analysis on day 7 post immunization and without CTV labeling of SWHEL B cells.

(A-E) Analysis of splenic SWHEL B cells and PC by flow cytometry. (A) Proportion of SWHEL B cells with a FAS+GL7+ GC phenotype, (B) SWHEL GC B cell count and (C) representation of IL-21R deficient cells among total SWHEL GC B cells. (D) CD98+ CD138+_SWHEL PC count and (E) representation of IL-21R deficient cells among total SWHEL PC. (F) Correlation of SWHEL GC B cell and PC count.

Data information: Data are representative of 6-10 biological replicates (n = 6-10) from two independent experiments. Data in B, D and F were analyzed after log transformation with with statistical analysis by Multiple paired t tests (A, B, D) and p-values corrected for multiple comparisons using Holm-Šídák method or One-way ANOVA with Tukey's post-test (C) or t test (E). Analysis in F using Pearson correlation coefficient. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Extended view figure legends
Asymmetric Distribution of Glucose Transporter mRNA Provides a Growth Advantage in Yeast (2019)
Figure 1

Figure 1 - The localization of both HXT2 mRNA and Hxt2 protein is cell-cycle dependent.

A The distribution of the GFP-tagged Hxt2 changes during the cell division. In small and medium budded cells (white arrowheads), Hxt2 is mainly present in the membrane of the mother cell. In large budded cells, Hxt2 is distributed over the entire plasma membrane (yellow arrowheads).

B HXT2 mRNA localization correlate with that of the protein. FISH with an HXT2-specific probe. Early in the cell cycle the mRNA is predominantly found in the mother cell (white arrowheads) while it is equally distributed after meta-to-anaphase transition (MAT) (yellow arrowheads).

C Quantification of (B). The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.

D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton. Cells were either treated for 15 min with 30 mg/ml latrunculin A or with 30 mg/ml benomyl. White arrowheads point to small-budded and yellow arrowheads to large budded cells.

E Quantification of (D) of three in dependent experiments with at least 50 cells each. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.

Data information: Scale bars in (A, B and D) correspond to 10 µm.
Asymmetric Distribution of Glucose Transporter mRNA Provides a Growth Advantage in Yeast (2019)
Figure 2

Figure 2 - Active translation is important for HXT2 mRNA release to the bud.

A, B Inhibiting translation by either applying Cycloheximide (CHX) or Verrucarin A (VerruA) leads to the retention of HXT2 mRNA in the mother cell after MAT. FISH of CHX or VerruA-treated cells. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.

C CHX treatment leads to increased transcript stability. Quantitative PCR of HXT2 mRNA in CHX-treated cells in comparison to prt1-1 mutant cells. The data represents 3 independent experiments and the standard deviation is given.

D Translation of HXT2 mRNA is more active late in the cell cycle. The HXT2 mRNA level bound to either di-/trisomes and polysomes (≥ 4 ribosomes) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1/S or M-phase followed by qPCR. At G1/S similar levels of the HXT2 mRNA were found in the different ribosome fractions, whereas in M-phase more mRNA was bound to polysomes, suggesting a more active translation in M-phase. A single experiment is shown. Two additional experiments are provided in Fig. EV1.

E The localization of HXT2 mRNA is independent of the Signal Recognition Particle (SRP). Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA.

Data information: Quantification of the FISH experiments in (A) and (E) was performed as in Fig. 1C. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. 150 cells per condition and cell-cycle stage from three independent experiments were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. ****: P < 0.0001 in a two-tailed, unpaired t-Test. Scale bars in (B) correspond to 5 µm.
Asymmetric Distribution of Glucose Transporter mRNA Provides a Growth Advantage in Yeast (2019)
Figure 3

Figure 3 - Loss of the Scp160/Bfr1/Asc1 complex and PKA cause enrichment of HXT2 mRNA in the bud after MAT.

A Deletion of SCP160, ASC1, or BFR1 increases HXT2 mRNA signal in the bud. FISH experiment with deletions in various RNA binding proteins.

B The effect of ∆scp160 on HXT2 mRNA localization is independent of its increased ploidy. Scp160 truncations that lack either the last two (∆C2) or the last four (∆C4) KH domains or a Tet-off SCP160 construct, in which expression is blocked by the addition of doxycycline (Doxy) confirm the phenotype observed in (A).

C Schematic depiction of the glucose responsiveness pathway.

D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud. Treatment of cells with forskolin (+Forsko) or using a ∆bcy1 strain, causes HXT2 mRNA to be bud-enriched.

E HXT2 mRNA enrichment upon refeeding is Ras2-dependent.

Data information: Quantification of FISH experiments in (A, B, D and E) was performed as in Fig. 1C. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. At least 150 cells per condition and cell-cycle stage from at least three independent experiments were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. ****: P < 0.0001; ***: P < 0.0005 in a two-tailed, unpaired t-Test.
Asymmetric Distribution of Glucose Transporter mRNA Provides a Growth Advantage in Yeast (2019)
Figure 4

Figure 4 - HXT mRNAs are retained in the mother before MAT, but only HXT2 mRNA is enriched in the bud after MAT upon refeeding.

A Table of the glucose affinity of the four most important hexose transporters.

B FISH of HXT1, 3 and 4 under glucose rich conditions. White arrowheads depict cells before, yellow arrowheads cells after MAT.

C Quantification of (B).

D Hyperactive PKA promotes enrichment of all HXTs after MAT.

E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation.

F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding. Daughters and mothers were separated through elutriation. qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions.

G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2-GFP transporter levels at the plasma membrane of the daughter cells. Daughter cells were enriched through elutriation. Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment. Western blot analysis confirms the higher amount of Hxt2-GFP transporter protein present in daughter cells under glucose-shift conditions

Data information: (C-E) Quantification of FISH experiments performed as described in Fig. 1C. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. At least 100 cells per condition and cell-cycle stage from at least three independent experiments were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. **** P < 0.0001 in a two-tailed, unpaired t-Test. (F) The mean of three independent experiments is plotted; bars represent standard deviation. *: P < 0.05 in a two-tailed, unpaired t-Test. (G) The mean of three independent experiments is plotted. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. **** P < 0.0001 in a two-tailed, unpaired t-Test. Scale bar in (B); 5 µm.
Asymmetric Distribution of Glucose Transporter mRNA Provides a Growth Advantage in Yeast (2019)
Figure 5

Figure 5 - HXT2 mRNA enrichment in the bud depends on Kar9 and nuclear segregation.

A RNA degradation is not essential for asymmetric HXT2 mRNA localization. FISH of wild-type and ∆xrn1 cells.

B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud. Cells were treated upon glucose addition with either 120 mg/ml benomyl for 15 min, 30 mg/ml latrunculin A for 30 min or 200 µM CK-666 for 30 min. HXT2 mRNA was detect by FISH.

C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function. HXT2 mRNA FISH with ∆bim1 and ∆kar9 strains. Arrows point to a nucleus with HXT2 signal.

D Quantification of the daughter cell enrichment from data displayed in (C).

E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in (C). Percentage of cells with asymmetric HXT2 mRNA staining is displayed.

Data information: Quantification of the FISH experiments in (A, B, and D) was performed as in Fig. 1C. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. At least 150 cells per condition from at least three independent experiments were analyzed. For the sample ∆kar9 with 2 nuclei/cell in (D), 50 cells were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. Quantification in (E) is based on three independent experiments. The bars represent standard deviation. *: P < 0.05, ****: P < 0.0001 in a two-tailed, unpaired t-Test. Scale bars in (C) correspond to 5 µm.
Asymmetric Distribution of Glucose Transporter mRNA Provides a Growth Advantage in Yeast (2019)
Figure 6

Figure 6 - NPC components aid the enrichment of HXT2 mRNA in the bud upon refeeding. A Deletion of NUP2 and MLP1/2 reduced HXT2 mRNA enrichment in the bud. FISH under glucose shift conditions with strains in which different NPC components were deleted.

B Quantification of (A).

C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift. FISH-IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84-HA. White arrows point to HXT2 mRNA signals on the nuclear envelope.

D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift. Stills of time lapse movies. HXT2 mRNA was visualized using the MS2-GFP system. Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus. Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus. Cyan: Nup84-mCherry; white arrowhead: HXT2 mRNA-MS2 + MCP-GFP.

Data information: Quantification in (B) was performed as in Fig. 1C. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. At least 175 cells per condition from at least three independent experiments were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. ****: P < 0.0001 in a two-tailed, unpaired t-Test. Scale bars in (A, C and D) correspond to 5 µm.
Asymmetric Distribution of Glucose Transporter mRNA Provides a Growth Advantage in Yeast (2019)
Figure 7

Figure 7 - HXT2 mRNA enrichment in the bud under glucose shift conditions provides a growth advantage to daughter cells.

A Schematic depiction of the growth assays. Cells expressing only one hexose transporter at a time were picked from a plate, serially diluted and plated on fresh YPD plates.

B HXT2 only strains grow faster than the other HXT only strain. Growth was assessed 2-3 days after incubation at 30°C.

C Workflow for the competition assay presented in (D). O/N cultures of HXT-only strains were diluted to the same OD600 and pooled. A sample was taken ('start'), the rest of the culture incubated O/N at 30°C. Cells were harvested ('end'), DNA was isolated from both samples, and the amount of HXT DNA was determined by qPCR.

D Quantification of competition assay. qPCR with primers against either HXT1, 2, 3 or 4 was performed. HXT gene abundance was normalized against actin.

E Strains that lack HXT2 grow slower when coming from quiescence compared to WT.

F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96% of cells independently of the cell cycle. Arrows point to examples of HXT2-U1A mRNA in the bud early and late in the cell cycle.

G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2.

H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers. Cells were re-fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points.

I Proposed model for the regulation of the asymmetric HXT2 mRNA distribution when cells come out of starvation or leave the quiescent state. For details, please see text.

Data information: (D) The results of three independent experiments are shown. Error bars represent standard deviation. Comparison of HXT-levels at the beginning and the end of growth shows that cells expressing only HXT2 outgrow other HXT-only cells in the range of 2 to 5-fold. *: P < 0.05 in a two-tailed, unpaired t-Test. (E) Data from three independent experiments are plotted. The bars represent standard deviation. (G) Data from four independent experiments were plotted. The bars represent standard deviation. A two-tailed unpaired t-test was performed for T = 4 hrs. *: P<0.05, **: P< 0.005. Quantification in (H) was performed as in Fig. 1C. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. At least 150 cells per condition from at least three independent experiments were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. ****: P < 0.0001 in a two-tailed, unpaired t-test.
HMCES safeguards replication from oxidative stress and ensures error-free repair (2020)
Figure 1

Figure 1. HMCES directly interacts with PCNA and is part of replisome complex.

(A) Summary of HMCES recovery upon TAP-MS analyses performed with 293T cells stably expressing SFB-PCNA. Cells were treated with CPT: camptothecin (1 μg/ml for 6 h); HU: hydroxyurea (2 mM for 16 h), UV: ultraviolet light irradiation (10 mJ/cm2 harvested after 6 h post-irradiation) or Thymidine (100 mM for 6 h). Recovered peptide-spectrum matches (PSMs) for PCNA and HMCES are indicated for each condition.

(B) Schematic representation of HMCES domain organization and deletion mutants used in this study.

(C) Immunoprecipitation (IP) of SFB-tagged full-length (FL) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction.

(D) GST-PCNA pulldown assay was used to evaluate the interaction between PCNA and full-length HMCES or indicated HMCES deletion mutants.

(E) aniPOND-Western blotting analysis revealed the presence of HMCES on replication fork. Cells were pulsed with EdU for 10 mins, and click-reaction was performed. In thymidine chase samples, cells were incubated with thymidine for another 30 mins prior to click-reaction.

(F) Analysis of HMCES level throughout the cell cycle. Cells were synchronized by double thymidine block and released for the indicated time points. CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases.