EMBO/soda-nel huggingface dataset:
hnRNP R negatively regulates transcription by modulating the association of P-TEFb with 7SK and BRD4 (2022)
Figure 5

Figure 5. Translation inhibition regulates RNA pol II phosphorylation through hnRNP R-dependent destabilization of CDK9

A, B. Western blot analysis of Ser2-phosphorylated and total RNA pol II levels in HNRNPR+/+ (A) and -/- (B) cells treated with DMSO or CHX for the indicated durations. GAPDH and α-Tubulin served as loading control.

C. Quantification of relative expression levels of Ser2-phosphorylated and total RNA pol II in (A, B). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; unpaired two-tailed t-tests (n = 4 biological replicates).

D, E. Western blot analysis of subcellular fractions from cells treated with DMSO or CHX for Calnexin, GAPDH and histone H3 (D), and Cyclin T1 and CDK9 (E). Cyt, cytosol; nuc+org, nuclear soluble proteins and organelles; chr, chromatin-associated proteins.

F. Quantification of relative expression of Cyclin T1 and CDK9 protein levels in subcellular fractions in (E). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; one-way ANOVA with Tukey's multiple comparisons test (n = 3 biological replicates).

G. Western blot analysis of Cyclin T1 levels in HNRNPR+/+ and -/- cells treated with DMSO or MG132. α-Tubulin served as loading control.

H. Quantification of relative expression levels of Cyclin T1 in (G). Data are mean with SD; ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).
hnRNP R negatively regulates transcription by modulating the association of P-TEFb with 7SK and BRD4 (2022)
Figure 6

Figure 6. Enhanced association of CDK9 with Cyclin K in hnRNP R knockout cells

A. TPM expression values for mRNAs encoding Cylin T1, T2 and K from RNA-seq data of wildtype HeLa cells (n = 4 biological replicates). Data are mean with SD (n = 4 biological replicates).

B. Western blot analysis of Cyclin K, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.

C. Western blot analysis of Cyclin K co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.

D. Quantification of Cyclin K co-immunoprecipitating with CDK9 in (C). Data are mean with SD; *P ≤ 0.05; unpaired two-tailed t-test (n = 3 biological replicates).
The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility (2021)
Figure 1

Figure 1. Two modes of CheY binding to the switch.

A, Run and tumble swimming modes in E. coli are regulated by CheY activation by phosphorylation or acetylation. The electron density map of the switch was produced by Thomas et al. (2006) and downloaded from http://dx.doi.org/10.1093/nar/gkv1126.

B, Scheme demonstrating the internalization of Atto647-labeled CheY(I95V) to single cells by electroporation.

C, Demonstration of single-molecule tracking in a single cell. Cell outline is shown as an ellipse. Left, Imaged FliM-YPet fluorescence. White dots indicate the estimated motor locations. White circles illustrate a 75 nm radius around these locations. Right, Contour map of the normalized sum of probabilities of the localization events of CheY(I95V)-Atto647 (from blue to red is low to high; black is zero probability). White circles show the motors' locations from the left panel.

D, Survival probabilities of unmodified CheY(I95V)-Atto647 at the switch (ΔcheA background; strain EW668). 63 cells were recorded with a total of 1316 trajectories in which CheY was found to interact with FliM. Note the logarithmic scale of the ordinate. Black line, a bi-exponential fit; dashed line, a fit of the fast decline process (see text for details).

E, as in (D) for acetylated CheY(I95V)-Atto647 (presence of acetate, 50 mM, pH 7.0; strain EW668). 56 cells were recorded with a total of 1904 trajectories.

F, As in (D) for phosphorylated CheY(I95V)-Atto647 (ΔcheZ background; strain EW669). 82 cells were recorded with a total of 2414 trajectories.

G, as in (D) for phosphorylated and acetylated CheY(I95V)-Atto647 (presence of acetate, 50 mM, pH 7.0; ΔcheZ background, strain EW669). 40 cells were recorded with a total of 2660 trajectories.

H, Experimental scheme of the FRET experiment. The attractant serine lowers the phosphorylation level of CheY. As a result, CheY dissociates from the switch and the energy transfer from YPet (conjugated to FliM) to mCherry (conjugated to CheY) is reduced.

I, CheY interacts with FliM∆N and this interaction is sensitive to chemotactic stimuli. Each curve is the mean of two FRET measurements of cells in response to an attractant stimulus (0.1 mM serine; in the case of FliMwt, a single measurement was performed with serine; another measurement of FliMwt with 1mM aspartate produced similar results). See Appendix Fig S1 for the details of FRET analysis. FRET ratio is the ratio of mCherry to YPet fluorescence. CheY-mCherry and mCherry concentrations were ~170 µM in the case of FliM∆N and ~15 µM in the case of FliMwt (Appendix Fig S2A for calibration of CheY concentration). Strains used: EW677 (FliMwt, red), EW659 (negative control of FliM∆N without CheY, purple), EW637 (FliM∆N, blue), and EW636 (FliM∆N ∆cheA, yellow).
The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility (2021)
Figure 2

Figure 2. FliM∆N motors respond differently to various modifications of CheY and FliMN-CheY.

A, Experimental scheme of flagellar motor tethering. The stub of sheared flagellum is tethered to glass by an anti-flagellin antibody (Silverman & Simon, 1974) in a flow chamber (Berg & Block, 1984).

B, Demonstration of rotation rate measured from a single cell; negative and positive values (below and above the blue bar) are counterclockwise and clockwise, respectively. The switch from negative to positive rotation rate at 3 s is shown in the montage to the right.

C, The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG), to positive stimuli. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM.

D, As in (C) for negative stimuli.

E, Response of tethered fliM∆N ΔcheA cells (strain EW634) to acetate (50 mM, pH 7.0) at different CheY concentrations. Lines and shaded regions are mean ± SEM.

F, Response of tethered fliM∆N ΔcheZ cells (strain EW635) to acetate. Details as in (E).

G, Contribution of acetylation and phosphorylation of CheY as well as its D13K mutation to clockwise rotation. The points are the mean clockwise rotation calculated from panels E and F at time segments 0-20 s and 160-180 s. The variants shown are fliM∆N ∆cheZ cells expressing CheY in the absence of presence of acetate (10 mM, pH 7.0) (i.e., CheY~P and CheY~P~Ac; blue and green curves; strain EW635), fliM∆N ∆cheA cells expressing CheY, in the absence of presence of acetate (CheY and CheY~Ac; purple and red, respectively; EW634), and fliM∆N ∆cheA cells expressing CheY(D13K) (burgundy; EW737). Each data point is the average of all experiments at a given CheY concentration, weighted by the sample number of each experiment. For data, see Table EV1. The CheY concentrations shown are estimates based on the calibration curves in Appendix Fig S2, for which a similar CheY expression system was used.

H, FliMN further activates CheY variants to produce clockwise rotation. The variants shown are fliM∆N ∆cheZ cells expressing FliMN-CheY (i.e., FliMN-CheY~P; blue curve; strain EW697), fliM∆N ∆cheA cells expressing FliMN-CheY, in the absence of presence of acetate (10 mM, pH 7.0) (FliMN-CheY and FliMN-CheY~Ac; purple and red, respectively; EW696), and fliM∆N ∆cheA cells expressing FliMN-CheY(D13K) (burgundy; EW739). See G for other details.

I, Response of fliM∆N ∆cheA cells expressing FliMN-CheY (strain EW696) to acetate and benzoate (10 mM each; pH 7.0). FliMN-CheY expression was induced with 800 μM IPTG. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The black arrow indicates the estimated time point at which the stimulus entered the flow chamber.
The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility (2021)
Figure 3

Figure 3. Biphasic switching of FliM∆N motors.

A, Dependence of clockwise rotation on the level of FliMN-CheY~Ac. The red and purple curves are two experimental days in which the clockwise production by fliM∆N ∆cheA cells expressing FliMN-CheY was measured as a function of FliMN-CheY concentration (strain EW696; these curves are partial data of the red curve in Figure 2H). Concentrations are estimates based on the calibration curves in Appendix Fig S2, for which a similar CheY expression system was used. Each data point is a mean ± SEM of the data shown in Table EV1 for the second and third experimental days of this strain (in the presence of acetate).

B, Response of the strain shown in (A) to addition and removal of varying acetate concentrations (pH 7.0). FliMN-CheY concentration was estimated to be ~300 μM. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrows indicate the estimated time points at which acetate entered or left the flow chamber. Black lines mark the linear part of the biphasic response to acetate removal. N is the number of cells.

C, Distribution of clockwise interval lengths at different FliMN-CheY~Ac concentrations in the strain shown in (A). FliMN-CheY was acetylated by acetate (10 mM, pH 7.0). The black lines illustrate the slopes of the short- and long-clockwise interval distributions. The color bar indicates the estimated FliMN-CheY~Ac concentration.

D, As in (C), but with distributions made with respect to the average clockwise levels of the cells.

E, Average clockwise interval length, calculated as the inverse of the fast (blue) and slow (red) rate constants from bi-exponential fits of the distributions in (D).
The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility (2021)
Figure 4

Figure 4. Impairment of FliMN-CheY binding to FliN results in long clockwise intervals.

A, Effects of FliMN-CheY acetylation and phosphorylation on clockwise rotation of tethered cells containing FliM∆N FliN(A93D) motors. Acetate concentration was 10 mM each (pH 7.0). FliMN-CheY and FliMN-CheY~P were produced by using ∆cheA and ∆cheZ backgrounds (strains EW713 and EW714), respectively. Each data point is the average of all measurements at a given FliMN-CheY concentration, weighted by the sample number of each experiment. The concentrations shown are estimates based on the calibration curves in Appendix Fig S2, for which a similar CheY expression system was used. Concentrations larger than 500 μM were calculated by the linear extrapolation of the calibration curve. For data see Table EV1.

B, Distribution of clockwise interval lengths at different average clockwise levels in ∆cheA cells containing FliM∆N FliN(A93D) motors and expressing FliMN-CheY (strain EW713), in the presence of acetate (10 mM, pH 7.0).

C, Average clockwise interval length, calculated as the inverse of the rate constants from mono-phasic fits of the distributions in (B).

D, Response of tethered ∆cheZ cells having FliM∆N FliN(A93D) motors to acetate addition and removal (red; strain EW714; FliMN-CheY concentration estimated at ~300 μM). Similar results were obtained with a ∆cheA strain (EW713). Tethered cells having FliM∆N FliNwt motors and containing FliMN-CheY (strain EW696) are shown for reference (blue; this is the orange curve in Fig 3B after cutting some time segments before and after acetate removal to synchronize with the other response). Acetate concentration was 10 mM (pH 7.0). The data shown are the mean ± SEM. N is the number of cells. Black lines indicate the clockwise-decay rates of FliNwt motors' slow phase and of FliN(A93D) motors following acetate removal.
The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility (2021)
Figure 5

Figure 5. Mutations designed to impair and enhance CheY binding to FliMM produce short- and long-clockwise intervals, respectively.

A, CheY crosslinks with FliM∆N-YPet in vivo. The cytoplasm of fliM∆N-YPet ΔcheZ cells overexpressing CheY (strain EW694) or FliMN-CheY (strain EW697) from a plasmid was crosslinked by glutaraldehyde and resolved by SDS-PAGE. 1, 2, 3 stand for three different experiments that underwent this procedure. The plus sign before ~14 and ~15 stands for +55 kDa of FliM∆N-YPet. Note that gel running suffered from parabolic distortion in band positions. The annotations relate to the lowest positions of the bands. The gel was imaged for FliM∆N-YPet fluorescence. To see the crosslinking products, the intense fluorescence of monomeric FliM∆N-YPet is shown at saturation. See Appendix Fig S6 for additional details.

B, Predicted binding interfaces of CheY (gray ribbon) and FliMM (green spheres) in E. coli. R94 of FliM is shown in blue. Part of the hydrophobic region of FliM (including E214) is shown in orange.

C, Clockwise rotation of tethered fliM(R94S)∆N ∆cheZ cells and fliM(R94L)∆N ∆cheZ cells expressing FliMN-CheY (strains EW731 and EW733, respectively) in the absence or presence of acetate (10 mM, pH 7.0). FliMN-CheY concentration was estimated to be ~4 mM by extrapolation of the calibration curves in Appendix Fig S2, for which a similar CheY expression system was used. No clockwise rotation was observed when benzoate substituted for acetate. Each data point is the mean of a separate experiment. Black line is the mean of all experiments. For sample number of each data point see Table EV1.

D, Clockwise rotation of tethered fliM(E214W)∆N ΔcheZ cells (strain EW718) at various concentrations of FliMN-CheY. The concentrations shown are estimates based on the calibration curve in Appendix Fig S2, for which a similar CheY expression system was used. Each data point is the average of all measurements at similar FliMN-CheY concentrations, weighted by the sample number of each experiment. The red points and curve are taken, as a reference, from Fig 2H. For data see Table EV1.

E, Distribution of clockwise intervals of fliM(R94SL)∆N ∆cheZ cells expressing FliMN-CheY (strains EW732 and EW734) in the presence of acetate (10 mM, pH 7).

F, Distribution of clockwise intervals of fliM(E214W)∆N ∆cheZ cells expressing FliMN-CheY (strain EW718) in the absence of acetate.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 1

Niche‐dependent Wnt activity in intestinal stem and progenitor cells

A Representative immunofluorescence staining of GFP of small intestinal tissue of LGR5‐GFP knock‐in (LGR5‐GFPki) mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.
B Quantification of GFP staining intensity of intestinal stem and progenitor cells (ISPCs) at indicated positions in basal crypts of LGR5‐GFPki mice (n = 80 crypts from four mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.
C Representative immunofluorescence staining of Msi1 of small intestinal tissue of young mTerc+/+ mice. Dashed line outlines a crypt. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.
D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc+/+ mice (n = 150 crypts from three mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.
E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5+ (GFP‐positive) cell gating.
F Representative FACS plots showing gating of LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations within the LGR5hi gating.
G–I mRNA expression of indicated genes relative to GAPDH in LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations (n = 3 mice). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 2

Suppression of Wnt signaling in intestinal basal crypts and ISPCs of aging telomerase‐deficient mice

mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway in freshly isolated small intestinal crypts of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 5 mice per group).
Axin2 mRNA expression in LGR5+ cells of 12‐ to 16‐month‐old G3 mTerc−/− mice and mTerc+/+ mice (n = 3 mice per group).
mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway and of the p53 target gene p21 in cultured crypts of 2‐month‐old G3 mTerc−/− mice and mTerc+/+ mice (n = 3 mice per group).

Data information: Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 3

Telomere dysfunction leads to preferential depletion of ISPCs with high Wnt activity at the crypt base

A–J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young (2–3 month) and old (12–16 month) LGR5‐GFPki, mTerc+/+ mice and LGR5‐GFPki, G3 mTerc−/− mice (n = 4 mice per group). (A, B, D, E, H, I) Representative FACS plots depicting the analysis of LGR5+ cells. Note the reduction in LGR5+ cells in G3 mTerc−/− mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo) and that within the fraction of LGR5hi cells, the cells with high LGR5‐reporter activity (LGR5hi‐high) are preferentially depleted in response to IR. (C, F) Quantification of (C) the number of LGR5+ cells and (F) the number of LGR5hi and LGR5lo cells. (G) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5+ cells. (J) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing old G3 mTerc−/− mice to mTerc+/+ mice.
K–O Immunofluorescence staining of GFP in basal crypts of 9‐month‐old LGR5‐GFPki, G3 mTerc−/− mice and age‐matched LGR5‐GFPki, mTerc+/+ mice (n = 2 mice per group). (K, L) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (M) Quantification of absolute number of GFP+ ISPCs at indicated positions at the crypt base per 50 crypts. (N) The histogram depicts the depletion rate (%) of LGR5+ cells in LGR5‐GFPki, G3 mTerc−/− mice compared to age‐matched LGR5‐GFPki, mTerc+/+ mice at indicated positions in the basal crypts. (O) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts. Note that the staining intensity of position 4 cells is equal in mTerc+/+ mice and G3 mTerc−/− mice and is significantly lower than position 1 and 2 cells of mTerc+/+ mice.

Data information: Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 4

Telomere dysfunction leads to preferential depletion of position 1 and 2 cells at the crypt bottom in between Paneth cells

A–I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4–5 mice per group). Representative pictures of (A, B) H&E staining, (D, E) PCNA staining, and (G, H) Msi1 staining are given. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (C) spindle‐shaped cells in between and adjacent to the Paneth cells, cells staining positive for (F) PCNA or (I) Msi1. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
J, K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9‐month‐old G3 mTerc−/− mice (K) and age‐matched mTerc+/+ (J) mice. Arrowheads point to positive cells. Dashed lines outline the crypts. Note that the remaining ISPCs in G3 mTerc−/− mice were mostly located in position 4 above the Paneth cells. Scale bar: 20 μm.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 5

γ‐irradiation leads to preferential depletion of ISPCs with high Wnt signaling activity

A–I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (A–F) Representative pictures of PCNA staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. Note the depletion of PCNA+ CBC cells in position 1 and 2 in between the Paneth cells at 3–48 h after IR and the regeneration of these cells at 4–6 days after IR. (G) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts at the indicated time points after IR. Mean values ± SEM are given. (H, I) Representative pictures of Olfm4 in situ hybridization. Arrowheads point to positive cells. Dashed lines outline the crypts in irradiated samples. Scale bar: 20 μm. Note the selective survival of ISPCs above the Paneth cells at 24 h after IR.
J–X Three‐month‐old LGR5‐GFPki, mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (J–N) Representative pictures of GFP staining at indicated time points after irradiation. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (O–X) Flow cytometry analysis of freshly isolated basal crypt cells at the indicated time points after IR (n = 4–10 mice per group). (O) The histogram depicts the number of LGR5+ cells in freshly isolated basal crypts at the indicated time point after IR. (P, Q) Flow cytometry analysis was used to determine the percentage of (P) LGR5hi cells and (Q) LGR5lo cells within the fraction of LGR5+ cells. (R‐W) Representative FACS plots of small intestinal crypt cells of non‐irradiated mice, 24 h after irradiation. Note the reduction in LGR5+ cell in irradiated mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo). (X) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing irradiated mice to non‐irradiated mice. Note the preferential depletion of LGR5hi‐high cells within LGR5hi cell population upon IR. Mean values ± SEM are given. NIR, non‐irradiated; IR, irradiated.

Data information: In (G, O–Q, X), unpaired two‐tailed Student's t‐test was applied.