EMBO/soda-nel huggingface dataset:
Centrosome function is critical during terminal erythroid differentiation (2022)
Figure 1

Figure 1: Ex vivo differentiation of Cdk5rap2null erythroblasts recapitulates key features of macrocytic anemia.

A Schematic of Cdk5rap2null (Cdk5rap2tm1b) allele generated from the EUCOMM knockout-first allele by Cre-mediated deletion of exon 5.

B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild-type (WT), heterozygous (HET) and null erythroid progenitors (EPs) isolated from fetal livers. Actin was used as loading control. ** indicates nonspecific band.

C Quantification of mean protein levels from (B). Numbers in brackets correspond to number of embryos analyzed.

D Immunofluorescence images of Cdk5rap2 WT, HET and null erythroid progenitors isolated from fetal livers. Progenitors were stained for CDK5RAP2 (grey), γ-tubulin (magenta) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 3 μm. Insets show higher magnification of centrosomes. Scale bar, 1 μm.

E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ-tubulin from (D). Numbers in brackets correspond to number of embryos analyzed with a total number of 470 (WT), 406 (HET) and 379 (null) progenitors.

F Complete blood count analysis from adult mice with genotypes as indicated. The number of mice analyzed is shown in brackets. RBC = red blood cell. MCV = mean corpuscular volume. RDW = red blood cell distribution width. HCT = hematocrit. MCH = mean corpuscular hemoglobin.

G Quantification of serum erythropoietin (EPO) levels from adult mice with genotypes as indicated. The number of mice analyzed is shown in brackets.

H Schematic of the ex vivo differentiation culture system.

I ImageStream images of ex vivo cultured enucleating EBs and reticulocytes. Cells were stained for TER119 (erythroid marker, green) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 5 μm.

J Quantification of enucleating EBs and reticulocytes after 48 hours (T48) in ex vivo culture. Genotypes are as indicated. The numbers in brackets correspond to the number of embryos analyzed.

K Quantification of enucleating EB size from (I). The numbers in brackets refer to the number of embryos analyzed.

Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (C, E, I and J) or number of mice (F and G). Statistical significances were determined by One-way ANOVA test with Tukey's (C, E, F, G, J) or Kruskal Wallis test with Dunn's (K) multiple comparisons.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
Centrosome function is critical during terminal erythroid differentiation (2022)
Figure 2

Figure 2: Cdk5rap2null erythroblasts undergo fewer divisions and enucleate prematurely.

A Deconvolved STED images of centrosomes in ex vivo cultured wild-type non-enucleating EBs (non-enucl.) or enucleating EBs/reticulocytes (enucl./RetiC). Cells were stained for γ-tubulin (magenta) and centriolar (CP110, CEP135) or PCM (CDK5RAP2, PCNT) proteins (cyan). Scale bar, 400 nm.

B Quantification of number of cell divisions after 24 (T24) and 48 (T48) hours. Ex vivo cultured erythroid progenitors with indicated genotypes were labelled with PKH26 to measure cell divisions. The numbers in brackets refer to the number of embryos analyzed.

C Schematic showing how ex vivo cultured erythroid progenitors were tracked in brightfield time-lapse microscopy experiments. Briefly, the two daughter cells produced by the first cytokinesis and their progeny were followed through terminal erythroid differentiation. Whenever a cell performed cytokinesis, enucleation or death, this was recorded as an event (ev). Examples depict paths taken by four daughter cells.

D Quantification of the frequency of cytokinesis, enucleation and death at each event (ev) in Cdk5rap2 wild-type or null erythroid progenitors as shown in (C). Four embryos for each genotype were analyzed.

E Quantification of cell cycle duration of Cdk5rap2 wild-type or null erythroid progenitors. The enucleation event was used as reference point to align previous divisions (see schematic in C). The number in brackets refer to the number of embryos analyzed.

Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Bar graphs display mean ± s.d. All statistical analysis was based on the number of embryos. Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test (B) or Mann-Whitney U-test (D and E).*P≤0.05, **P≤0.01.
Centrosome function is critical during terminal erythroid differentiation (2022)
Figure 4

Figure 4: Centrosome depletion closely phenocopies impact of CDK5RAP2 loss on late-stage erythroblasts.

A Schematic shows experimental outline of Centrinone-B (CB) treatment of ex vivo cultured erythroid progenitors. CB treatment results in centrosome loss over multiple divisions as depicted on the right.

B Immunofluorescence images of untreated (untx) or CB-treated erythroid progenitors at stated differentiation stages after 24 (T24) and 48 (T48) hours in ex vivo culture. Cells were stained for γ-tubulin (magenta), TER119 (erythroid marker, green) and DNA (Hoechst, blue). Scale bar, 3 μm.

C Quantification of centrosome numbers (marked by γ-tubulin) upon CB treatment from (B). For the left panel, in total 632 (untx, T24), 637 (CB, T24), 544 (untx, T48) and 467 (CB, T48) cells from two litters were analyzed. T24 = 24 hours. T48 = 48 hours. For the right panel, in total 799 (untx, non-enucl), 142 (untx, enucl), 235 (untx, RetiC), 839 (CB, non-enucl), 127 (CB, enucl) and 131 (CB, RetiC) cells from two litters were analysed.

D Quantification of enucleating EBs and reticulocytes at T48 of ex vivo culture following CB treatment. Number of litters analyzed is shown in brackets.

E Quantification of cell cycle profile of enucleating EBs at T48 of ex vivo culture following CB treatment. EBs were pulsed with BrdU according to Fig 3A. Number of embryos analyzed is shown in brackets. See Fig EV3I for cell cycle profile of TER119pos cells.

F, G Quantification of cell (F) and nuclear (G) size at T48 in all enucleating EBs (left panels in F and G) or according to their DNA content (right panels in F and G) following CB treatment of ex vivo culture. Percentage of enucleating EBs in each category from (E) is shown below x-axis. Number of embryos analyzed is shown in brackets.

H Quantification of number of cell divisions at T48 of ex vivo culture following CB treatment. Ex vivo cultured cells were labelled with the membrane dye PKH26 to monitor cell divisions. Number of embryos analyzed is shown in brackets.

I Quantification of number of cell divisions at T48 performed as in (H) following CB treatment of ex vivo culture. Left panel shows all enucleating EBs, whereas right panel shows enucleating EBs according to their DNA content. Percentage of enucleating EBs in each category is shown below the x-axis. Number of embryos analyzed is shown in brackets.

Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C and D) or number of embryos (E-H). Statistical significance was determined by Mann-Whitney test (D-H and I left) or One-way ANOVA with Tukey's multiple comparisons test (I right). *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
Centrosome function is critical during terminal erythroid differentiation (2022)
Figure 5

Figure 5: Mitotic spindle assembly is severely impaired in erythroblasts when centrosomes or CDK5RAP2 are absent

A Immunofluorescence images of untreated (untx) or centrinone-B (CB)-treated cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), phospho-Histone H3 (pHH3, green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm.

B Quantification of mitotic spindle morphologies in untreated (untx) or centrinone-B (CB)-treated cells at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. In total 315 (untx, T24), 367 (CB, T24), 295 (untx, T36), 282 (CB, T36) cells were analyzed from 3 litters.

C Immunofluorescence images of Cdk5rap2HET and Cdk5rap2null cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), pHH3 (green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm.

D Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. At T24, 3 Cdk5rap2WT (323 cells), 3 Cdk5rap2HET (296 cells) and 2 Cdk5rap2null (197 cells) embryos were analyzed. At T36, 5 Cdk5rap2WT (387 cells), 6 Cdk5rap2HET (384 cells) and 3 Cdk5rap2null (202 cells) embryos were analyzed.

E Schematic (left) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points. Representative ImageStream images of ex vivo cultured cells at different mitotic stages (right). Cells were stained for phospho-Histone H3 (pHH3, green), TER119 (erythroid marker, magenta) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 10 μm.

F and G Quantification of pHH3pos, TER119pos cells and mitotic stages (see text for details) after 36 hours (T36) upon DMSO or nocodazole treatment according to (E) using ImageStream. Number of embryos analyzed is shown in brackets.

Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (B) or number of embryos (D, F and G). Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test (B, D, F and G).*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
Centrosome function is critical during terminal erythroid differentiation (2022)
Figure 6

Figure 6: Erythroblasts, unlike MEFs, show no sign of centrosome maturation.

A Quantification of mean centrosomal signal intensities of PCM proteins in wild-type MEFs. MEF lines (n=5; independently derived) were analyzed with a total of 168 (CDK5RAP2), 158 (CEP192), 164 (PCNT) and 332 (γ-tubulin) interphase cells and 59 (CDK5RAP2), 68 (CEP192), 61 (PCNT) and 120 (γ-tubulin) mitotic cells. Note that the same CDK5RAP2 data also forms part of Fig EV1F.

B Immunofluorescence images of cells in interphase (I) and mitosis (M) at 24 (T24) and 36 (T36) hours of ex vivo culture. Cells were stained for γ-tubulin (magenta), protein of interest (POI, CDK5RAP2, PCNT or CEP192 in grey) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm.

C Quantification of mean centrosomal signal intensities of PCM proteins from (B). Three litters were analyzed with the following number of cells for I, T24; M, T24; I, T36 and M, T36: 290, 157, 259 and 174 (CDK5RAP2); 291, 190, 203 and 181 (PCNT); 314, 122, 283 and 152 (CEP192); 895, 465, 720 and 493 (γ-tubulin).

D Quantification of mean centrosomal PCNT signal intensities during mitosis at 24 (T24) and 36 (T36) hours of ex vivo culture. Genotypes are as indicated. Number of embryos analyzed is shown in brackets. 227 (WT), 282 (HET) and 158 (null) cells were analyzed for T24 and 370 (WT), 348 (HET) and 196 (null) cells were analyzed for T36.

E Quantification of mean γ-tubulin signal intensities at interphase or mitotic centrosomes after 36 hours (T36) in ex vivo culture. Genotypes are as indicated. Number of embryos analyzed is shown in brackets. 326 (WT), 243 (HET) and 195 (null) interphase cells and 292 (WT), 281 (HET) and 299 (null) mitotic cells were analyzed. See Fig EV6B for representative immunofluorescence images.

F Quantification of mean centrosomal signal intensities of γ-tubulin from (EV1E). Numbers in brackets correspond to number of MEF lines analyzed with 168 (WT) and 154 (null) interphase cells and 59 (WT) and 56 (null) mitotic cells.

G Quantification of PCM foci numbers in CB-treated mitotic erythroid progenitors after 24 (T24) and 36 (T36) hours of ex vivo culture. Three litters were analyzed with the following total number of cells for untx, T24; CB, T24; untx, T36 and CB, T36: 141, 221, 174 and 150 (CDK5RAP2); 112, 122, 116 and 85 (PCNT); 165, 183, 145 and 106 (CEP192); 403, 527, 440 and 411 (γ-tubulin). See Fig EV5B for representative immunofluorescence images.

Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of MEF lines (A and F) number of litters (C and G) or number of embryos (D-E). Statistical significance was determined by Mann-Whitney test (A) or One-way ANOVA with Tukey's multiple comparisons test (C-G).*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
Centrosome function is critical during terminal erythroid differentiation (2022)
Figure 7

Figure 7: Elevated TP53 activity resulting from centrosome or CDK5RAP2 loss is not responsible for abnormal erythroblast differentiation.

A Immunoblot analysis of TP53, P27 and P21 levels in ex vivo cultured Cdk5rap2 wild-type (WT), heterozygous (HET) and null EBs after 24 (T24) and 48 (T48) hours. GAPDH was used as loading control. ** indicates unspecific band.

B Quantification of mean protein levels from (A). Numbers in brackets represent number of embryos analyzed.

C Quantification of cell cycle profiles of ex vivo cultured Trp53HET or Trp53null enucleating EBs with or without CB treatment. EBs were pulsed with BrdU according to Fig 3A. Numbers in brackets represent number of embryos analyzed. See Fig EV6C for cell cycle profiles of TER119pos cells.

D Quantification of ex vivo cultured Trp53HET or Trp53null enucleating EBs and reticulocytes after 48 hours (T48) upon CB treatment. Numbers in brackets represent number of embryos analyzed.

E Quantification of cell cycle profiles of ex vivo cultured enucleating EBs with the indicated genotypes after 48 hours (T48). Number of embryos analyzed is shown in brackets. See Fig EV6F for cell cycle profiles of TER119pos cells.

F Quantification of ex vivo cultured enucleating EBs and reticulocytes with the indicated genotypes after 48 hours (T48). Number of embryos analyzed is shown in brackets.

G Model shows the origin of EBs enucleating with 2N versus 4N DNA content and the consequences on reticulocyte production.

Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). All statistical analysis were based on number of embryos. All statistical significances were determined by One-way ANOVA with Tukey's multiple comparisons test.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

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MiR-9 modulates and predicts the response to radiotherapy and EGFR-inhibition in HNSCC (2021)
Figure 2

Figure 2. miR-9 expression is induced by EGFR pathway activation in HNSCC.

A. Graph reporting the normalized Luciferase activity of miR-9 promoter, expressed as fold over the untreated condition, in FaDu cells serum starved and then stimulated with 10% Fetal Bovine Serum (FBS) for up to two hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and one-way ANOVA test was used to verify the statistical significance.

B. qRT-PCR analyses of normalized miR-9 expression, expressed as fold over the untreated condition, in FaDu cells serum starved and then stimulated with 20 ng/ml of Epidermal growth factor (EGF) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and one-way ANOVA test was used to verify the statistical significance.

C. Graph reporting the normalized Luciferase activity of miR-9 promoter, expressed as fold over the untreated condition, in FaDu cells treated with Gefitinib (GEFI) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and one-way ANOVA test was used to verify the statistical significance.

D. qRT-PCR analyses of normalized miR-9 expression, expressed as fold over the untreated condition, in control (shCTR) and antimiR-9 FaDu cells treated with Gefitinib (GEFI) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA test was used to verify the statistical significance.

E. Graph reporting the normalized Luciferase activity of miR-9 promoter, expressed as fold over the untreated condition, in FaDu cells treated with Cetuximab (CTX) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and one-way ANOVA test was used to verify the statistical significance.

F. qRT-PCR analyses of normalized miR-9 expression, expressed as fold over the untreated condition, in control (shCTR) and antimiR-9 FaDu cells treated with Cetuximab (CTX) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA test was used to verify the statistical significance.

G/H. Graph reporting the cell viability of control (shCTR) and antimiR-9 FaDu cells treated with increasing concentration of Gefitinib (G) or Cetuximab (CTX) (H) as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and unpaired t-test was used to verify the statistical significance per each dose.

I. Dot plot reporting the correlation of EGFR and miR-9 expression in primary HNSCC samples evaluated by qRT-PCR. The number of analyzed samples (n), the Spearman correlation value (r) and its significance (p) are reported in the graph.

In the figure A.U. = Arbitrary Units *p<0.05; ***p<0.001.
MiR-9 modulates and predicts the response to radiotherapy and EGFR-inhibition in HNSCC (2021)
Figure 5

Figure 5. miR-9 protects HNSCC cells from RT-induced cell death.

A. Graph reporting cell viability of control (shCTR) and antimiR-9 FaDu cells treated with increasing concentration of the indicated drugs for 72 hours and analyzed using the MTS cell viability assay. Data represent the mean (±SD) of three independent experiments each performed in sextuplicate and unpaired t test was used to calculate the statistical significance per each dose.

B. Clonogenic assay of control (shCTR) and antimiR-9 FaDu cells not irradiated (NIR) or treated with 2 or 5Gy IR. On the left, typical images of cell clones are shown. On the right the graph reports the percentage (±SD) of survived cells respect to not irradiate cells in three independent experiments performed in triplicate. Unpaired t test was used to calculate the statistical significance per each dose.

C. Left, WB analyses of the indicated protein expression in control (shCTR) and antimiR-9 FaDu cells not irradiated (NIR) or treated with 5Gy IR and allowed to repair for the indicated hours (h). Tubulin was used as loading control. On the right, protein quantification analyses of KLF5 and Sp1 expression normalized on tubulin loading control. On the right, graphs report the quantification of the indicated proteins normalized on tubulin expression. Data are expressed as mean (±SD) of three independent experiments and unpaired t test was used to calculate the statistical significance at time point.

D. Left panel reports typical immunofluorescence images of control (shCTR) or antimiR-9 FaDu cells, not irradiated (NIR) and analyzed 1, 4 or 8 hours after 2Gy IR (γH2AX green, pS10-H3 red, nuclei in blue). Graphs report the percentage of γH2AX (middle) and pS10-H3 (right) positive cells. Data represent the mean (±SD) of three independent experiments in which at least 10 randomly selected fields were evaluated. Unpaired t test was used to calculate the statistical significance at each time point.

E. Graphs report the qRT-PCR analyses evaluating the expression of miR-9 (left) or SP1 (right) in cells treated as in C. Data are the mean (±SD) of three independent experiment performed in duplicate. Two-way ANOVA with Sidak's multiple comparison test was used to calculate the statistical significance.

F. Graph reporting cell viability of control (shCTR) and antimiR-9 FaDu cells, overexpressing or not SP1, and treated with increasing concentration of Bleomycin (BLEO) for 72 hours and analyzed using the MTS cell viability assay. Data represent the mean (±SD) of three independent experiments each performed in sextuplicate. Two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance. *p<0.05; **p<0.01; ***p<0.001.

In the figure A.U. = Arbitrary Units; *p<0.05; **p<0.01; ***p<0.001.
MiR-9 modulates and predicts the response to radiotherapy and EGFR-inhibition in HNSCC (2021)
Figure 6

Figure 6. miR-9 expression regulates and predicts the response to RT+Cetuximab combination therapy.

A. Schema of the in vivo analyses of tumor growth in mice (n=10/group) injected with FaDu cells. After tumor appearance, mice were injected intratumoral with high titer viruses encoding for control or antimiR-9 sequences. After two injections of virus mice were treated with Cetuximab (IP injections) and RT (4Gy dose) as indicated and then sacrificed 36 days after injections.

B. Graph reports the tumor volume of tumors described in A (n=10 mice/group). Data represent the mean (±SD) and two-way ANOVA was used to verify the statistical significance.

C/D. Typical images of Hematoxylin&Eosin (H&E) (C) and Ki67 expression (Ki67) (D) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in A/B.

E. Graph reports the percentage of Ki67 positive cells in tumors, as described in D. Data are expressed as mean (±SD) of Ki67 percentage counted in five randomly selected fields per tumor, in at least 4 tumors per group. Two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.

F. TUNEL assay performed in tumors described in C. On the left, typical immunofluorescence images (blue - nuclei, green - TUNEL). On the right, graph reports the percentage of TUNEL positive cells in tumors as described in C. Each dot represents a tumor. Data represent the mean (±SD) and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.

G. Kaplan-Mayer curve evaluating the overall survival of HNSCC patients treated with RT+Cetuximab (CTX) combination included in the TCGA dataset, segregated on the expression of miR-9 in the primary tumor (Low expression <75819 reads n=8; High expression ≥75819 reads n=23). Number of evaluated samples (n) and p value are reported in the graph. Statistical significance was calculated with long-rank test.

H. Kaplan-Mayer curve evaluating the progression free survival of HNSCC patients treated with RT+Cetuximab (CTX) combination at the CRO-Aviano National Cancer Institute and at the University Cattolica segregated based on miR-9 expression in primary tumors, defined as the expression in above (high expression n=18) or below (low expression n=17) the median expression, as defined by ddPCR. Hazard Ratio (HR) and statistical significance were calculated with long-rank (Mentel-Cox) test and are reported in the graph.

In the figure *p<0.05; **p<0.01; ***p<0.001.

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