Targeting cyclin-dependent kinases for the treatment of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease with poor prognosis and limited therapeutic options. We screened for pathways that may be responsible for the abnormal phenotype of pulmonary arterial smooth muscle cells (PASMCs), a major contributor of PAH pathobiology, and identified cyclin-dependent kinases (CDKs) as overactivated kinases in specimens derived from patients with idiopathic PAH. This increased CDK activity is confirmed at the level of mRNA and protein expression in human and experimental PAH, respectively. Specific CDK inhibition by dinaciclib and palbociclib decreases PASMC proliferation via cell cycle arrest and interference with the downstream CDK-Rb (retinoblastoma protein)-E2F signaling pathway. In two experimental models of PAH (i.e., monocrotaline and Su5416/hypoxia treated rats) palbociclib reverses the elevated right ventricular systolic pressure, reduces right heart hypertrophy, restores the cardiac index, and reduces pulmonary vascular remodeling. These results demonstrate that inhibition of CDKs by palbociclib may be a therapeutic strategy in PAH.

5. The effects of the CDK inhibitors either can't be determined -too toxic -or are very modest. Here, off-target effects of the inhibitors must be shown by histology of other tissues, which are also affected by PH in humans and the animal models. In certain instances, blood chemistries should also be provided. It's not enough to just show that the CDKs are expressed in other tissues in a normal animal model. There is some impetus to explain why one of the drugs, dinaciclib was not tolerated while the other drug, which has an upstream target, is better tolerated. Is this due to the model (MCT), PH in general, or effect on another organ system? 6. For Figure 1B, please provide a densitometry of p-ERK/ERK graph. Although there is more p-ERK in PAH cells, there is also more total ERK. Also, why aren't there two bands in the control samples? Was the same antibody used? 7. The in vivo studies report very limited hemodynamic (systemic and right heart) data and the echocardiograms present very limited data. Full systemic and right heart hemodynamics needs to be added (i.e., heart rate, arterial blood pressure, SVR, etc.) and standard information such as weights, including pre-model, time of introduction of CDK inhibitor, and end of study.
Reviewer #2 (Remarks to the Author): In this manuscript the Authors focused on pulmonary arterial hypertension (PAH). By performing a kinome profiling of human pulmonary arterial smooth muscle cells (PAMSC) derived from healthy individuals and patients affected by idiopatic PAH (IPAH), the Authors identified cyclin-dependent kinases (CDKs) as being overactivated in IPAH-PAMSC. They then investigated the effect of the treatment of PAMSC with the CDK inhibitors dinaciclib and palbociclib, on the cell cycle. Finally the Authors investigated the potential therapeutic efficacy of the CDK4 and CDK6 inhibitor palbociclib in a PAH rat model. The topic is interesting, however I have several concerns as following described.
Major points - Figure 1b. The Authors state that in order to demonstrate a different phenotype of human pulmonary arterial smooth muscle cells (HPASMC) derived from individuals with idiopathic pulmonary arterial hypertension (IPAH-HPASMC) versus HPASMC derived from healthy individuals, they investigated the activation of ERK1/2 signaling pathway. It is not clear why the ERK1/2 signaling pathway was selected, the Authors should explain better the rationale of this choice. In addition, assuming that the numbers "#1", "#2" and "#3" refer to individual patients, this aspect should be clarified in the figure legend.
-The Authors affirm that HPASMC derived from IPAH patients are hyperproliferating cells, however they do not show any characterization of these cells in terms of proliferation potential in comparison with HPASMC derived from healthy individuals.
- Figure 1c-e. The Authors state that the CDK activity is up-regulated in IPAH patients-derived HPASMC ( fig. 1c) when compared to HPASMC derived from healthy individuals. However, the data relative to the activity of CDKs are not clearly shown. The resolution of the figures 1d and 1e is low and the text in the figures is not readable. -The Authors investigated the expression of CDK2, CDK4 and CDK6 in human tissues derived from healthy and IPAH patients. However, the Authors do not clearly explain the reason why they focused on these three CDKs. -The expression of CDK2, CDK4 and CDK6 on mRNA level (figure 3d) and protein level (figure 3e) is not consistent. For example, the brain and muscle tissues showed the highest mRNA levels of CDK4 when compared to the other organs, however CDK4 was undetectable on protein level in both brain and muscle. The Authors should comment on this aspect. -In figure 4 and in figure 5a-c the Authors show the effect of the treatment with dinaciclib and palbociclib on cell proliferation, DNA synthesis etc, however it is not clear whether these experiments were performed by using HPASMC derived from healthy or IPAH patients. The Authors should clarify this aspect. Also, the Authors did not show any data relative to the measure of the IC50, for both the drugs, in their system.
-In addition, it is not clear why the Authors used as control the cells treated with basal media (BM) and not with GM-2 media as the cells treated with dinaciclib and palbociclib.
-In figure 5a and 5b the Authors show the effect of the treatment of the cells with dinaciclib and palbociclib on the cell cycle and cell apoptosis. In lines 195-197, the Authors state that there was no sign of cell death induction upon treatment of the cells with the drugs, since the fraction of cells in SubG1 phase did not increase (figure 5a-b). However, in lines 200-204 the Authors affirmed that dinaciclib triggered apoptosis-mediated cell death (figure c). The Authors should explain this discrepancy. It is opinion of this reviewer that a deeper characterization of the apoptosis process, also under a molecular point of view (i.e. by investigation the potential involvement of caspases), would be necessary.
-In figure 5d the Authors show the effect of the treatment of healthy HPASMCs on the levels of phosphorylated and total Rb. Here, both dinaciclib and palbociclib reduced the levels of phosphorylated Rb (pRb), without affecting the levels of total Rb. By contrast, in the western blot shown in figure 5e, the levels of both pRb and total Rb were reduced in healthy HPASMCs treated with both dinaciclib and palbociclib. The Authors should explain this discrepancy. Also, concerning the levels of p21, the Authors do not comment on the reported differences between cells from healthy and pathological individuals or among cells treated with the different drugs. Finally, in figure  5d, the Authors show the detection of PCNA, which is not mentioned neither in the manuscript nor in the figure legend.
-In the experiments shown in figure 5e the Authors introduced the use of imatinib, however they do not explain the rationale of using this drug and of comparing it to dinaciclib and palbociclib.
-It is opinion of this reviewer that the levels of phosphorylation of CDK2, CDK4, CDK6 and CDK9 and their total levels should be included in the western blot analysis. Indeed, since these CDKs are the targets of the used drugs, their characterization is crucial. -In figure 7i and 7j, the Authors show that the treatment of MCT-treated rats with palbociclib led to a reduction in the mRNA levels of CCN2A and CDK1. From these data the Authors concluded (lines 535-534) that "cellular proliferation was almost completely blocked in the animals treated with palbociclib, as evidenced by the reduced levels of two important transcriptional CDK-Rb-E2F downstream target genes, CCN2A and CDK1". It is opinion of this reviewer that the observed reduction in the mRNA levels of CCN2A and CDK1 is not enough to conclude that cell proliferation was blocked. Additional experiments, such as the immunohistochemical detection of proliferation markers like ki67 should be performed. Also, an evidence that palbociclib actually affected the activity of the targeted CDK4 and CDK6 in vivo should be included. The levels of phosphorylated and total CDK4 and CDK6 should be determined (for example by immunohistochemistry).
Minor points -Line 219, please correct "transcriptional target genes of activated by E2F" with "transcriptional target genes of E2F".
-In the Results section, lines 98-107, the Authors explained in a really detailed manner the experimental procedure followed for the acquisition of the data shown in figure 1c-1e. Maybe this part can be moved to the Material and Method section.   (c) Bar chart displaying the overall prediction of kinases with higher activity in IPAH-patient derived samples. Normalized kinase statistics is a mathematical based algorithm indicating the estimated relative kinase activity while the specificity score reflects the reliability and accuracy of the prediction.

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This kind of investigation does not allow for a comprehensive signaling network presentation which could only be done on the level of the whole phospho-proteome. Detailed explanation of the technique has been discussed in the revised version of the manuscript in the supplement section (page 56-57, line 1167-1193). Next, we addressed the point of heterogeneity within the samples of each group e.g. healthy and IPAH, by physically performing another data assessment, increasing the sample size to n=6 with subsequent analysis of the data. The individual data sets are included in the supplementary material (figure S1; revised).
3 Figure S1. Heat map from the peptide micro array obtained from all individual samples. Raw data for all individual HPASMC samples from both entities presented as a heat map with log-transformed fluorescence signals due to kinase-mediated substrate serine/threonine phosphorylation. Based on those signal intensities an upstream kinase activity was predicted with increased levels for distinct CDKs. Despite a variation between the different individual healthy HPASMCs, as well as in the IPAH-patient derived cells, increased CDK activity is a common feature of all IPAH-HPASMCs. In the revised version, we decided to present a heat map showing the mean phosphorylation pattern of multiple samples for the two groups (figure 1a, b; revised).   Based on the above-mentioned analysis, we focused on CDKs as these data sets clearly demonstrates CDK6, CDK9, CDK4 and CDK2 with a highest prediction value in IPAHderived HPASMCs (figure 1c; revised). For better clarity, we (i) changed the format of the two group comparison description, (ii) included the computational upstream kinase prediction in the revised version of the manuscript, and (iii) provided a phosphorylation profile for upstream kinase prediction of CDK2, CDK4, CDK6, and CDK9 (figure 1d; revised). Although the signals are not homogenous and display distinct patterns of phosphorylation, we still identified increased CDK activity as a common feature for all IPAH-HPASMCs. By this, our former statement in lines 117-119 has been clarified.  , and CDK6 (c), with corresponding IHC for P-Rb protein and its downstream target gene and common proliferation marker PCNA (proliferation cell nuclear antigen). Cellular identity was visualized by antibodies against α-SMA (alpha-smooth muscle actin) and vWF (von-Willebrand factor). Images were taken at 400-fold magnification. Scale bar 20 µm.
Results referring to this question can be found in the revised version of the manuscript (page 7-8, line 157-165). Furthermore, to assess the influence of palbociclib on structural remodeling in experimental models of PH, we performed double staining with anti-α-actin and anti-vWF, followed by quantification of the degree of muscularization of small peripheral pulmonary arteries. Notably, palbociclib treatment in two experimental models of PH (MCT and Su/Hox) markedly reduced the number of fully muscularized arteries (figure 6a, 6b, 8a, 8b; revised) along with reduced number of PCNA positive cells (figure 6c and 8d; revised), confirming that palbociclib induces cell cycle arrest and subsequently improves structural vascular remodeling. The degree of muscularization of small pulmonary arteries (diameter 25 -50 µm) was determined ex vivo via immunhistological staining of lung sections for vWF (brown) and α-SMA (violet) together with methylgreen for counterstaining. M: fully muscularized; P: partially muscularized; N: non-muscularized. Representative images for all three study groups are shown. (b) Medial wall thickness of vessels with a diameter of 20-50 µm was determined by Elastica-van-Gieson staining and is presented as percentage. Data are presented as mean±SEM and statistical analysis was performed using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons; ** p < 0.01 for MCT+Palbo versus MCT; § p < 0.05, § § p < 0.01 for MCT+Palbo versus healthy; ### p < 0.001 for healthy versus MCT. The medial wall thickness as well as the corresponding ratio of neointima/media of the depicted study groups were determined by Elastica-van-Gieson staining. Data are presented as mean±SEM and statistical analysis was performed using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons; ** p < 0.01, *** p < 0.001 for MCT+Palbo versus MCT; § p < 0.05, § § p < 0.01, § § § p < 0.001 for MCT+Palbo versus healthy; ### p < 0.001 for healthy versus MCT.     Echocardiograms were recorded at baseline, 21 days after Su5416 administration at the beginning of hypoxia exposure and again on day 35 after a 14-day period of treatment with 75 mg/kg body weight palbociclib (or placebo) daily by gavage under re-exposure to normoxic conditions. (b-l) On day 35, hemodynamics and cardiac function were assessed in vivo 14 days after treatment with with palbociclib (Su/Hox+Palbo, n=7) (under hypoxia exposure). Similarly, normoxic rats (n=8) and animals injected with Su5416 and placebo (under hypoxia exposure) (Su/Hox, n=8) were used as control groups. Data are presented as mean±SEM and statistical analysis was performed using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001 for Su/Hox+Palbo versus Su/Hox; § < 0.05, § § p < 0.01, § § § p < 0.001 for Su/Hox+Palbo versus Nox; # < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 for Nox versus Su/Hox. Table 1: Further hemodynamic description of both experimental PAH models. Data are presented as mean±SD and statistical analysis was performed using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons (a) MCT rat model; § p < 0.05, § § § § p < 0.0001 for post-versus pre-treatment of MCT; # p < 0.05, ## p < 0.01, ### p < 0.001 for posttreatment MCT+Palbociclib versus MCT.
Further assessment of structural remodeling suggests that palbociclib not only reduces muscularization of distal pulmonary arteries, but also neointima formation, arguing its influence on both hyper-proliferative smooth muscle cells and endothelial cells (     HPAECs. Cells were synchronized in endothelial specific basal media (ECBM supplemented with 0.5% FCS) and then treated with the indicated concentrations of dinaciclib or palbociclib or DMSO as control in the presence of growth media (ECGM) for 24 h. IC 50 graphs determining the half maximal inhibitory concentration of dinaciclib (a) and palbociclib (b) to block EC-GM induced proliferation of healthy HPAECs as determined by BrdU incorporation (relative absorbance [A 370nm -A 492nm ], n=1). IC 50 values were calculated from measurements of one primary cell isolate (run in two independently performed triplicates) by the non-linear regression (curve fit) module with a variable slope using four parameters as provided by GraphPad Prism software. Representative microscopic images from distinct cell culture conditions (c) were taken with a 400-fold magnification prior the performance of representative Western blots (d) for the detailed analyses of cellular signaling on protein level after 24 hours of treatment with dinaciclib, palbociclib, or staurosporine (SSP) in healthy HPAECs with regard to CDK activation and downstream Rb-E2F pathway induction. CCNA2 (left) and CDK1 (right) mRNA expression (normalized to GAPDH as a house keeping gene) of healthy HPAECs (e) upon 24 hours of inhibitor exposure. All data from one primary cell isolates (run twice in triplicates each) are presented as mean±SEM of the n-fold change (2 -∆∆Ct ) compared with DMSO treated control samples (black bar). Statistical analysis was performed using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons; *** p < 0.001. P-values for distinct conditions are only given for their comparison with DMSO-treated control cells (black bar), and the significance of the difference between the three conditions with various concentrations of each of the CDK inhibitors is represented as follows: § p < 0.05, § § p < 0.01, § § § p < 0.001. R5: We agree with the reviewer that models of PH can contribute to the toxic effects and no animal model per se completely recapitulates human PAH vascular pathologies. Particularly, monocrotaline via reactive metabolites (e.g. dehydromonocrotaline) was shown to influence several organs apart from pulmonary vasculature and can induce myocarditis, liver injury, kidney injury and even muscle impairment [3][4][5] . Although we strongly believe that the MCT model of PH is a valuable model to assess the therapeutic effects of anti-remodeling compounds on the pulmonary vasculature, the toxic effects of MCT on other organs questions the interpretation of toxic /off-target effects of any compound in this model. In contrast, Su5416 in combination with hypoxia (Su/Hox) predominantly influences the pulmonary vasculature, making it as a suitable model to assess also the potential toxicity of the drugs. Therefore, we thank the reviewer for suggesting us to address the therapeutic efficacy of palbociclib in the Su/Hox rat model. Importantly, we found that palbociclib treatment significantly reduced RVSP, PVRI and improved cardiac performance (see R3). A pathologist blinded to the experimental groups, evaluated histological or structural abnormalities in Su/Hox+palbociclib or placebo treated rat organs (left ventricle, liver, kidney and intestine) and could not find evidence for potential toxicity of palbociclib (figure S9; revised).     , n=5). Similarly, healthy rats (Healthy, n=6) and animals injected with MCT and placebo (MCT, n=5) were used as control groups. Data are presented as mean±SEM and statistical analysis was performed using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001 for MCT+Palbo versus MCT; § < 0.05, § § p < 0.01, § § § p < 0.001, § § § § p < 0.0001 for MCT+Palbo versus healthy; # < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 for healthy versus MCT. recorded at baseline, 21 days after Su5416 administration at the beginning of hypoxia exposure and again on day 35 after a 14-day period of treatment with 75 mg/kg body weight palbociclib (or placebo) daily by gavage under re-exposure to normoxic conditions. (b-l) On day 35, hemodynamics and cardiac function were assessed in vivo 14 days after treatment with with palbociclib (Su/Hox+Palbo, n=7) (under hypoxia exposure). Similarly, normoxic rats (n=8) and animals injected with Su5416 and placebo (under hypoxia exposure) (Su/Hox, n=8) were used as control groups. Data are presented as mean±SEM and statistical analysis was performed using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001 for Su/Hox+Palbo versus Su/Hox; § < 0.05, § § p < 0.01, § § § p < 0.001 for Su/Hox+Palbo versus Nox; # < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 for Nox versus Su/Hox.    Raw data for all individual HPASMC samples from both entities presented as a heat map with log-transformed fluorescence signals due to kinase-mediated substrate serine/threonine phosphorylation. Based on those signal intensities an upstream kinase activity was predicted with increased levels for distinct CDKs.

-The Authors affirm that HPASMC derived from IPAH patients are hyper-proliferating cells, however they do not show any characterization of these cells in terms of proliferation potential in comparison with HPASMC derived from healthy individuals.
R: Previous publications from our group and others have shown that HPASMCs isolated from IPAH-patients have a higher proliferative potential 1,2 . Since growth factor stimulation will lead to activation of kinases and their respective substrates, we decided to study the kinome    R: In order to address the increased CDK activity in IPAH-HPASMCs, we decided to present a heat map showing the mean phosphorylation pattern of multiple samples for the two groups (figure 1a, b; revised) in the revised version of the manuscript. The n-number has been increased to n=6 and all individual data sets are included in the supplementary material (figure S1; revised). An increased CDK activity is a common feature of all IPAH-HPASMCs.
We now also added the detailed results from the computational upstream kinase prediction with further kinases of potential interest in the form of a bar chart (figure 1c; revised). Bar chart displaying the overall prediction of kinases with higher activity in IPAH-patient derived samples. Normalized kinase statistics is a mathematical based algorithm indicating the estimated relative kinase activity while the specificity score reflects the reliability and accuracy of the prediction.

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Based on the above-mentioned analysis, we focused on CDKs as these data sets clearly demonstrates CDK6, CDK9, CDK4 and CDK2 with a highest prediction value in IPAHderived HPASMCs (figure 1d; revised).  29 R: We would like to thank the reviewer for this comment and the observation that several CDKs have been found to be active in our kinase screen. Since, altered cell cycle and an increased proliferative potential of IPAH-HPASMCs 2,6 is the major driver of pulmonary vascular remodeling and CDK1, CDK2, CDK4 and CDK6 are considered as bona fide cell cycle regulators, we focused on them 7 . Importantly, among these cell cycle regulating CDKs, computational upstream kinase prediction displayed a highest prediction value of CDK2, CDK4 and CDK6 (but not of CDK1) in IPAH-derived HPASMCs ( figure 1c; revised), instigating us to focus on CDK2, CDK4 and CDK6.  miR-6883-5p and miR-149*. These microRNAs directly bind to 3′UTR of CDKs and repress its translation 8,9 . Thus, the regulation of CDK activity by specific miRNAs might be important both under physiological and pathological conditions. Thus, we concomitantly analyzed protein expression of CDK2/4/6/9 in all experimental settings ( figure 1c, 3e-f, 6d, 8e, S5d,   S9c).     . Representative Western blots for the detailed analyses of cellular signaling on protein level after 24 hours of treatment with dinaciclib, palbociclib, or staurosporine (SSP) in healthy HPASMCs (e) and IPAH-HPASMCs (f) with regard to CDK activation and downstream Rb-E2F pathway induction. (g) Western blot analysis for (P-)Rb and Cyclin D3 of healthy and IPAH-PASMCs treated with dinaciclib, palbociclib, or imatinib. CCNA2 (left) and CDK1 (right) mRNA expression (normalized to GAPDH as a house keeping gene) of healthy HPASMCs (h) and diseased IPAH-HPASMCs (i) upon 24 hours of inhibitor exposure. All data from two individual primary cell isolates (run twice in triplicates each) are presented as mean±SEM of the n-fold change (2 -∆∆Ct ) compared with DMSO treated control samples (black bar). Statistical analysis was performed using one-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons; *** p < 0.001. P-values for distinct conditions are only given for their comparison with DMSO-treated control cells (black bar), and the significance of the difference between the three conditions with various concentrations of each of the CDK inhibitors is represented as follows: § p < 0.05, § § p < 0.01, § § § p < 0.001.
We removed p21 as it is not a direct target gene of the CDK signaling pathway and included the PCNA data in the result and discussion section as well as in the figure legend. R: Imatinib is a well-known PDGFR/Abl/tyrosine kinase inhibitor which was the first kinase inhibitor that demonstrated anti-prolifetive and anti-remodeling efficacy in experimental and clinical PAH 12,13 . The rationale of using imatinib in this context is to compare the mode of action and underlying downstream molecular mechanisms of both CDK inhibitors. While we observed that both CDK inhibitors completely blocked Rb expression and activation in healthy-and IPAH-HPASMCs, imatinib did not appear to exert its known anti-proliferative effects by interfering with the Rb-E2F signaling pathway, suggesting the specific mode of action of CDK inhibitors. Due to extensive data obtained during revision process, we rearranged the figures and moved this data to another section (figure 3g; revised). R: We appreciate this comment and addressed this concern accordingly. We performed additional Western blots to detect P-CDK2, P-CDK6, and P-CDK9 on protein level from (i)            R: We will make sure, that all figures and labels are at appropriate size. Fig. 2 id much appreciated and is very clear with respect to expression of the CDKs in the vasculature of human iPAH. The interesting finding that expression of CDK2 and CDK4 seems to be highest in the adventitial cells while CDK 6 appears to be highest in the endothelium. Expression of these CDKs doesn't appear to be much greater in iPAH vs controls. Since you perform the kinase profiling studies with PASMCs, this suggests that either increased expression of the CDKs isn't important and/or PASMC CDK activity is activated by mediators from other cell types.

Q1. The new data in
This apparent disconnect should be acknowledged.

R1:
We appreciate the thorough evaluation of our IHC stainings of human specimens. We agree with the reviewer that the intensity of CDK2 and CDK4 immunoreactivity seems to be highest in the adventitial cells while CDK 6 appears to be highest in the endothelium and immunoreactivity of CDKs is not strikingly altered in IPAH. However, interestingly (i) direct targets of CDKs such as p-Rb and PCNA immunoreactivity is strongly increased in all vascular cells of IPAH.
(ii) peptide-based kinase activity profiling reveals increased activity of the CDK-Rb-E2F signaling pathway in PASMCs from IPAH patients.
(iii) prominent expression of CDKs was observed in human PASMCs and a significant change in the pattern of phosphorylation of CDKs was noted when comparing the healthy PASMCs and IPAH-PASMCs.
(iv) dinaciclib and palbociclib, two CDK activity inhibitors employed in this study, decreased the proliferation of both PASMCs and PAECs.  R4: We were intensively searching for adequate antibodies for all CDKs in this study and had difficulties to find antibodies specifically detecting the phosphorylated forms for Western blot analysis. Due to high sequence homology it is almost impossible to generate antibodies that clearly distinguish the different isoforms. We tested several of them using several techniques for immunohistological stainings of human and rat lung specimen. Unfortunately, we could not find any phospho-specific antibodies (i.e. for P-CDK2 and P-CDK6) which resulted in reliable and trustworthy data. In particular for P-CDK4, no antibody was available at the time of our analysis and until today, only one antibody from Mybiosource (#MBS9126724) or LSBio (LS-C411605) for Western blot analysis of human P-CDK4 with an unknown performance (that means quality and specificity) for other applications and species can be obtained. For this reason, we focused on Western blot analyses of the direct downstream targets P-Rb and PCNA as appropriate readouts to evaluate the inhibitory effects of dinaciclib and palbociclib on CDK activity.