Wouldn’t you like to spend less money on your reagents?

We all have certain consumer buying habits. But we also know that sometimes we do not necessarily get the best products at the most price-attractive conditions, if we simply buy the same products from the same source again and again…

What is true for our private behavior, most probably applies to our habits when it comes to buying research reagents for our laboratory. [Read more…]

Differentiate between K48- and K63-specific Ubiquitination

Ubiquitination is a common post translational protein modification that occurs through an isopeptide linkage between the C-terminus of ubiquitin and the ε-amino group of a lysine residue on the target substrate. Ubiquitin itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can participate in further ubiquitination, generating poly-Ub chains. The ability of Ubiquitin to form polymers through various lysines as well as its NH2-terminus appears to be central to the versatility of this system in regulating a variety of cellular processes. [Read more…]

Trapping PARP-DNA complexes – anti-cancer drug screening

PARPs (Poly ADP ribose polymerases) are found in the nucleus of the cell and they are involved in SSB repair (single-strand DNA breaks). PARP is known to bind damaged DNA through its N-terminal zinc finger domain. Subsequently it starts to synthesize a poly (ADP-ribose) chain which serves as a signal for other DNA-repairing enzymes.

SYNTHETIC_ LEATHALITY

Fig. 1: Principle of synthetic lethality

PARP inhibitors are considered to be promising  candidates as anti cancer drugs (recently Olaparib, the first drug directed against PARP1, has been approved by the European commission). One of the reasons is that some tumors are more dependent on PARP than regular cells. These cancer cells are mutated in BRCA1 or BRCA2 – both genes which are involved in key DNA damage repair mechanisms. In healthy cells PARPs can function as a kind of back-up system and let the cells survive even without functional BRCA gene products. When PARPs are inhibited the cells do not possess any functional SSB repair mechanism anymore and are bound to die (Fig. 1; take a look at PARPs as cancer drug targets – first EC-approved drug to learn more about the concept of “synthetic lethality”). [Read more…]

Modulating or inhibiting Caspase activities

In a previous post, I discussed Caspases as pharmaceutical targets – how to screen for inhibitors?

Today I would like to concentrate on Caspase inhibitors/modulators, which allow for in-depth characterisation of your enzyme of interest and which can serve as reference compounds in caspase inhibitor screenings. A short recap: Caspases (Cysteine-dependent aspartate-directed proteases) belong to the family of cysteine proteases and are involved in networks controlling cell death (apoptosis and necrosis) and inflammation. Amongst the 12 known human caspases, 5 have been described as playing a crucial role in  apoptosis (Caspase-3, -6, -7, -8, and -9), 4 have been linked to processes in inflammation (Caspase-1, -4, -5, and -12), and 3 (Caspase-2, -10, and -14) could not yet be exactly classified concerning their functions. [Read more…]

Differentiate between Apoptosis, Necroptosis, Autophagy & Ferroptosis

Are you looking for “Get your ELISA results before lunch! ” by Ana Arratzio? click here

Cell death can be caused by external factors such as infection or trauma – a process which is call necrosis. On the other hand, cell death can be mediated by intracellular programs – in this cases we talk about programmed cell death.

What are the different types of programmed cell death?

When we talk about programmed cell death we usually think of…

Apoptosis

Fotolia_71744424_XSThe induction of apoptosis leads to characteristic cell changes and finally to death (see How to measure early apoptotic events). These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Furthermore, changes affecting the membrane, nucleus, cytoplasm, and mitochondria occur. Apoptosis involves a complex cascade of reactions regulated by specific proteases called caspases (see Caspases as pharmaceutical targets – how to screen for inhibitors?), and results in DNA degradation. Apoptotic processes have been researched in an extensive variety of diseases. Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.

Another quite prominent type of programmed cell death is represented by…

Autophagy

Autophagy process - Blog ThumbnailAutophagy describes the fundamental catabolic mechanism during which cells degrade dysfunctional and unnecessary cellular components (see How to manipulate and measure Autophagy). This process is driven by the action of lysosomes and promotes survival during starvation periods, as the cellular energy level can thus be maintained. During autophagy autophagosomes are generated, organelles which are surrounded by double membranes and which contain the cellular components to be degraded. The autophagosome formation is induced by class 3 phosphoinositide-3-kinase, Atg 6 (autophagy-related gene 6) and ubiquitin or ubiquitin-like modifications of the target proteins. Subsequently, autophagosomes traffick through the cytosol of the cell and finally fuse with lysosomes to form an autolysome. After this fusion the cargo is degraded by lysosomal hydrolases. Excessive autophagy leads  to cell death – a process which can be morphologically differentiated from apoptosis.

But there are less known types of programmed cell death, such as…

FerroptosisFerrostatin-0 - Andrea 11

This process is triggered by an iron-dependent accumulation of lethal ROS in cells. It can be induced by e.g. erastin which blocks the cellular uptake of cystine and thus blocking the intracellular antioxidant defense mechanism by limiting the production of intracellular glutathione (GSH), the primary cellular antioxid. ROS generation is iron-dependent as its accumulation and cell death can be supressed by the iron chelator deferoxamine (see Ferroptosis – and the way to inhibit it).

And finally…

Necroptosis

Necroptosis is indeed a programmed form of necrosis. As in all forms of necrotic processes, cells break open and leak their contents into the intercellular environment. In contrast to necrosis, leaking of the membrane during necroptosis is regulated by the cell. Necroptosis has been well described as a non-apoptotic “cellular suicide” process which represents a viral defense mechanism. Moreover, it plays a role in inflammatory diseases such as pancreatitis and Crohn’s disease.

Tools to differentiate between Apoptosis, Necroptosis, Autophagy, and Ferroptosis

Focus Biomolecules recently launched a set of compounds which are know to selectively inhibit one of the four programmed cell death types described above.

If you are interested to confirm a hypothesis that cells are undergoing a certain response due to the “hypothesized” mechanism – these inhibitors can be of great help to you!

Ferrostatin 1 - structure

Ferrostatin-1

  • Ferrostatin-1  – a specific inhibitor of ferroptosis. Ferrostain-1 has been shown to control lipid ROS (reactive oxygen species)
  • 3-Methyladenine – a specific autophagy inhibitor
  • Necrostatin-1 – a specific inhibitor of necroptosis
  • Z-VAD-FMK – one of a series of well defined apoptosis modulators (you’ll find here an overview about apoptosis modulators)

Any questions or comments? Please use the form below!

 

 

Ferroptosis – and the way to inhibit it

A lot of compounds are known and available to modulate (inhibit or activate) the most prominent mode of programmed cell death – Apoptosis. In a recent post, I summarized methods to detect early Apoptosis and at the end of this post you’ll find a list of apoptosis modulators (available through tebu-bio). But there are other modes of programmed cell death, such as Autophagy (see How to manipulate and measure Autophagy), Necroptosis, and Ferroptosis.

Today I would like to focus on Ferroptosis, and invite you to take a look at the first Ferroptosis inhibitor on the market. [Read more…]

How to measure early apoptotic events?

Apoptosis and cellular apoptotic events. Source: tebu-bio

Fig. 1: Process of Apoptosis

Apoptosis is the most prominent process of programmed cell death (for an overview see Fig. 1). Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Furthermore, changes affecting the membrane, nucleus, cytoplasm, and mitochondria occur. Apoptosis involves a complex cascade of reactions regulated by specific proteases called caspases (take a look at previous posts on Caspases as pharmaceutical targets – how to screen for inhibitors?), and results in DNA degradation. Apoptotic processes have been researched in an extensive variety of diseases. Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.

Besides apoptosis other types of programed cell death are known, such as autophagy (see How to manipulate and measure Autophagy?), necroptosis, and ferroptosis (look out for an imminent post I’ll be doing about this iron-dependent form of cell death very shortly, as well as tools to differentiate between apoptois, necroptosis, autophagy, and ferroptosis).

In this post, let’s take a look at methods and kits allowing to measure early apoptotic events. [Read more…]

How to detect active caspase in cells?

In a recent post I gave an overview about the role of Caspases in different human diseases and introduced tools to measure Caspase activity and inhibition by compounds in homogenous biochemical assays: Caspases as pharmaceutical targets – how to screen for inhibitors? Today I would like to give you an overview about a method and kits which allow to detect active Caspases in cells and which give an insight into the apoptotic status of the respective cells. Hence the effects of inducers of apoptosis can be investigated in living cells. [Read more…]

Caspases as pharmaceutical targets – screening for inhibitors?

Caspases (cysteine-dependent aspartate-directed proteases) belong to the family of cysteine proteases and are involved in networks controlling cell death (apoptosis and necrosis) and inflammation. 12 human caspases have been described so far (1.). Human Caspases have been classified according to their roles in apoptosis (Caspase-3, -6, -7, -8, and -9) and inflammation (Caspase-1, -4, -5, and -12). Caspase-2, -10, and -14 can be less easily classified concerning the function (for an overview see 2.).

So let’s take a further look at their role, and some of the tools available to investigate and screen compounds modifying Caspase activities.

[Read more…]

Staining Actin and Tubulin – from WB to Live Cell Imaging

Anti Anti Ab - IF photo

Fig. 1: Immunofluorescence images of mouse Swiss 3T3 cells stained with anti-actin antibody (027AAN01).

Actin and Tubulin, as the major cytoskeleton structures, are crucial components of a plethora of processes in cell biology. Both are very much involved in stabilizing the cell shape, and especially in cell movements (e.g. cell migration) and intracellular movements and transport mechanisms.

Thus visualizing Actin and Tubulin in fixed or living cells and detecting them in biological samples (e.g. in Western Blots) belongs to basic experimental set-ups in Cell Biology.

A broad range of tools is available for all kind of experimental levels, in this post we’ll take a look at some of these you can use from Western Blot to Live Cell Imaging. [Read more…]